Tag library compounds, compositions, kits and methods of use

ABSTRACT

Families of compositions are provided as labels, referred to as eTag reporters for attaching to polymeric compounds and assaying based on release of the eTag reporters from the polymeric compound and separation and detection. For oligonucleotides, the eTag reporters are synthesized at the end of the oligonucleotide by using phosphite or phosphate chemistry, whereby mass-modifying regions, charge-modifying regions and detectable regions are added sequentially to produce the eTag labeled reporters. By using small building blocks and varying their combination large numbers of different eTag reporters can be readily produced attached to a binding compound specific for the target compound of interest for identification. Protocols are used that release the eTag reporter when the target compound is present in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuing patent application ofapplication Ser. No. 09/602,586, filed Jun. 21, 2000, which is acontinuing application of application Ser. No. 09/561,579, filed Apr.28, 2000.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of this invention is separable compositions for use inmultiplexed assay detection.

[0004] 2. Background of the Invention

[0005] As the human genome is elucidated, there will be numerousopportunities for performing assays to determine the presence ofspecific sequences, distinguishing between alleles in homozygotes andheterozygotes, determining the presence of mutations, evaluatingcellular expression patterns, etc. In many of these cases one will wishto determine in a single reaction, a number of different characteristicsof the same sample. Also, there will be an interest in determining thepresence of one or more pathogens, their antibiotic resistance genes,genetic subtype and the like.

[0006] In many assays, there will be an interest in determining thepresence of specific sequences, whether genomic, synthetic or cDNA.These sequences may be associated particularly with genes, regulatorysequences, repeats, multimeric regions, expression patterns, and thelike

[0007] There is and will continue to be comparisons of the sequences ofdifferent individuals. It is believed that there will be about onepolymorphism per 1,000 bases, so that one may anticipate that there willbe an extensive number of differences between individuals. By singlenucleotide polymorphism (snp's) is intended that there will be aprevalent nucleotide at the site, with one or more of the remainingbases being present in substantially smaller percent of the population.

[0008] For the most part, the snp's will be in non-coding regions,primarily between genes, but will also be present in exons and introns.In addition, the great proportion of the snp's will not affect thephenotype of the individual, but will clearly affect the genotype. Thesnp's have a number of properties of interest. Since the snp's will beinherited, individual snp's and/or snp patterns may be related togenetic defects, such as deletions, insertions and mutations involvingone or more bases in genes. Rather than isolating and sequencing thetarget gene, it will be sufficient to identify the snp's involved.

[0009] In addition, the snp's may be used in forensic medicine toidentify individuals. While other genetic markers are available, thelarge number of snp's and their extensive distribution in thechromosomes, make the snp's an attractive target. Also, by determining aplurality of snp's associated with a specific phenotype, one may use thesnp pattern as an indication of the phenotype, rather than requiring adetermination of the genes associated with the phenotype.

[0010] The need to determine many analytes or nucleic acid sequences(for example multiple pathogens or multiple genes or multiple geneticvariants) in blood or other biological fluids has become increasinglyapparent in many branches of medicine. The need to study differentialexpression of multiple genes to determine toxicologically-relevantoutcomes or the need to screen transfused blood for viral contaminantswith high sensitivity is clearly evident.

[0011] Thus most multi-analyte assays or assays which detect multiplenucleic acid sequences involve mutiple steps, have poor sensitivity andpoor dynamic range (2 to 100-fold differences in concentration of theanalytes is determined) and some require sophisticated instrumentation.

[0012] Some of the known classical methods for multianalyte assaysinclude the following:

[0013] a. The use of two different radioisotope labels to distinguishtwo different analytes.

[0014] b. The use of two or more different fluorescent labels todistinguish two or more analytes.

[0015] c. The use of lanthanide chelates where both lifetime andwavelength are used to distinguish two or more analytes.

[0016] d. The use of fluorescent and chemiluminescent labels todistinguish two or more analytes.

[0017] e. The use of two different enzymes to distinguish two or moreanalytes.

[0018] f. The use of enzyme and acridinium esters to distinguish two ormore analytes.

[0019] g. Spatial resolution of different analytes, for example, onarrays to identify and quantify multiple analytes.

[0020] h. The use of acridinium ester labels where lifetime or dioxetaneformation is used to quantify two different viral targets.

[0021] Thus an assay that has higher sensitivity, large dynamic range(10³ to 10⁴-fold differences in target levels), greater degree ofmultiplexing, and fewer and more stable reagents would increase thesimplicity and reliability of multianalyte assays.

[0022] The need to identify and quantify a large number of bases orsequences potentially distributed over centimorgans of DNA offers amajor challenge. Any method should be accurate, reasonably economical inlimiting the amount of reagents required and providing for a singleassay, which allows for differentiation of the different snp's ordifferentiation and quantitation of multiple genes.

[0023] Finally, while nucleic acid sequences provide extreme diversityfor situations that may be of biological or other interest, there areother types of compounds, such as proteins in proteomics that may alsooffer opportunities for multiplexed determinations.

BRIEF DESCRIPTION OF RELATED ART

[0024] Holland (Proc. Natl. Acad. Sci. USA (1991)88:7276) discloses theexonuclease activity of the thermostable enzyme Thermus aquaticus DNApolymerase in PCR amplification to generate specific detectable signalconcomitantly with amplification.

[0025] The TaqMan assay is discussed by Lee in Nucleic Acid Research(1993)21:16 3761).

[0026] White (Trends Biotechnology (1996)14(12):478-483) discusses theproblems of multiplexing in the TaqMan® assay.

[0027] Marino, Electrophoresis (1996)17:1499 describeslow-stringency-sequence specific PCR (LSSP-PCR). A PCR amplifiedsequence is subjected to single primer amplification under conditions oflow stringency to produce a range of different length amplicons.Different patterns are obtained when there are differences in sequence.The patterns are unique to an individual and of possible value foridentity testing.

[0028] Single strand conformational polymorphism (SSCP) yields similarresults. In this method the PCR amplified DNA is denatured and sequencedependent conformations of the single strands are detected by theirdiffering rates of migration during gel electrophoresis. As withLSSP-PCR above, different patterns are obtained that signal differencesin sequence. However, neither LSSP-PCR nor SSCP gives specific sequenceinformation and both depend on the questionable assumption that any basethat is changed in a sequence will give rise to a conformational changethat can be detected.

[0029] Pastinen, Clin. Chem. (1996)42:1391 amplifies the target DNA andimmobilizes the amplicons. Multiple primers are then allowed tohybridize to sites 3′ and contiguous to a snp (“single nucleotidepolymorphism”) site of interest. Each primer has a different size thatserves as a code. The hybridized primers are extended by one base usinga fluorescently labeled dideoxynucleoside triphosphate. The size of eachof the fluorescent products that is produced, determined by gelelectrophoresis, indicates the sequence and, thus, the location of thesnp. The identity of the base at the snp site is defined by thetriphosphate that is used. A similar approach is taken by Haff, NucleicAcids Res. (1997)25:3749 except that the sizing is carried out by massspectroscopy and thus avoids the need for a label. However, both methodshave the serious limitation that screening for a large number of siteswill require large, very pure primers that can have troublesomesecondary structures and be very expensive to synthesize.

[0030] Hacia, Nat. Genet. (1996)14:441 uses a high-density array ofoligonucleotides. Labeled DNA samples are allowed to bind to 96,60020-base oligonucleotides and the binding patterns produced fromdifferent individuals were compared. The method is attractive in thatSNP's can be directly identified, but the cost of the arrays is high andnon-specific hybridization may confound the accuracy of the geneticinformation.

[0031] Fan (Oct. 6-8, 1997 IBC, Annapolis Md.) has reported results of alarge scale screening of human sequence-tagged sites. The accuracy ofsingle nucleotide polymorphism screening was determined by conventionalABI resequencing.

[0032] Allele specific oligonucleotide hybridization along with massspectroscopy has been discussed by Ross in Anal. Chem. (1997)69:4197.

[0033] Holland, et al., PNAS USA (1991)88, 7276-7280, describes use ofDNA polymerase 5′-3′ exonuclease activity for detection of PCR products.

[0034] U.S. Pat. No.5,807,682 describes probe compositions for detectinga plurality of nucleic acid targets.

SUMMARY OF THE INVENTION

[0035] Compounds and methods are provided for multiplexed determinationsaffording convenient separation of released identifying tags based onindividual physical, properties of the tags. The methods can beperformed in a single vessel and may involve a plurality of reagentsadded simultaneously or consecutively. In one group of embodiments, masswill be involved in the characteristic allowing for separation. Onegroup of identifying tags for electrokinetic analysis is characterizedby having regions, which serve as (1) a cleavable linking region; (2) amass-modifying region; (3) a charge-modifying region: and (4) adetectable region, the number of different regions depending in part onthe method of separation and identification. Compounds that have thesedistinctive regions find use in conjunction with other compounds wherethe regions are combined in the same moiety. Of particular interest isthe use of building blocks for forming the compounds, where thesynthesis is performed in a repetitive manner using the same linkingchemistry at a plurality of stages. The subject compounds are linked tobinding compounds for identification to provide identifying reagents,where binding of an identifying reagent target in an assay systemresults in the release of the identifying tag (hereinafter referred toas an, “eTag™ reporter”) where the eTag reporters can be differentiated.Large numbers of eTag reporters can be provided in kits comprising alinking functionality for bonding to the binding compounds or kits ofbuilding blocks can be provided for synthesizing eTag reporters in situin conjunction with the synthesis of the binding compound. Of particularinterest is the use of the subject eTag reporters in identification ofnucleic acids and proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1A shows the design and synthesis of eTag reporters on solidphase support using standard phosphoramidite coupling chemistry.

[0037]FIG. 1B illustrates separation of eTag reporters designed topossess unique charge to mass ratios.

[0038]FIG. 1C shows charge modifier phosphoramidites. (EC or CE iscyanoethyl).

[0039]FIG. 1D shows polyhydroxylated charge modifier phosphoramidites.

[0040] FIGS. 2A-2I show structures of different eTag reporters.

[0041]FIG. 3 is a schematic illustrating exemplary high voltageconfigurations utilized in a CE² LabCard™ device during an enzyme assay.

[0042]FIG. 4 is two electropherograms demonstrating eTag reporteranalysis using a CE² LabCard. The figure shows the separation ofpurified labeled aminodextran with and without sensitizer beads. Theaddition of the sensitizer beads lead to the release of the eTagreporter from the aminodextran using singlet oxygen produced bysensitizer upon the irradiation at 680 nm. Experimental conditions:Separation buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO; voltageconfigurations as shown in FIG. 3; assay mixture had 29 μg/mlstreptavidin coated sensitizer beads and irradiated for 1 min at 680 nmusing 680±10 nm filter and a 150 W lamp.

[0043]FIG. 5 is multiple electropherogams demonstrating eTag reporteranalysis using a CE² LabCard. The figure shows the separation ofpurified labeled aminodextran that has been irradiated for differentlengths of time. Experimental conditions: Separation buffer 20.0 mMHEPES pH=7.4, and 0.5% PEO; voltage configurations as shown in FIG. 3;assay mixture had 27 Ag/ml streptavidin coated sensitizer beads andirradiated at 680 nm using 680±10 nm filter and a 150 W lamp.

[0044]FIG. 6 is multiple electropherograms demonstrating eTag reporteranalysis using a CE² LabCard. The figure shows the separation ofpurified labeled aminodextran using different concentrations ofsensitizer beads. The higher concentration of sensitizer beads leads tothe higher release of eTag reporters from the labeled aminodextran.Experimental conditions: Separation buffer 20.0 mM HEPES pH=7.4, and0.5% PEO; voltage configurations as shown in FIG. 3; assay mixture wasirradiated for 1 min at 680 nm using 680±10 nm filter and a 150 W lamp.

[0045]FIG. 7 depicts the linear calibration curve for the release ofeTag reporters as a function of the sensitizer bead concentration.Results were obtained using a CE² LabCard. Experimental conditions:Separation buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO; voltageconfigurations as shown in FIG. 3; assay mixture was irradiated for 1min at 680 nm using 680±10 nm filter and a 150 W lamp.

[0046]FIG. 8 is a data curve showing the effect of the concentration oflabeled aminodextran on the eTag reporter release, demonstrated in thisfigure, the lower concentration of labeled aminodextran for a givenconcentration of sensitizer beads leads to more efficient eTag reporterrelease (or higher ratio of eTag reporter released to the amount oflabeled aminodextran). Results were obtained using a CE² LabCard.Experimental conditions: Separation buffer 20.0 mM HEPES pH=7.4, and0.5% PEO; voltage configurations as shown in FIG. 3; assay mixture had29 μg/ml of sensitizer beads and was irradiated for 1 min at 680 nmusing 680 +10 nm filter and a 150 W lamp.

[0047]FIG. 9 is multiple electropherograms showing separation ofindividual eTAG reporters. The figure illustrates obtainable resolutionof the reporters which are identified by their ACLA numbers.

[0048]FIG. 10 is multiple electropherograms showing a separation on a310 analyzer that has occurred after an amplification reaction, in thepresence of probe and primer without the addition of avidin.

[0049]FIG. 11 is multiple electropherograms showing a separation on a310 analyzer that has occurred after an amplification reaction, in thepresence of probe and primer with the addition of avidin.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0050] Methods and compounds are provided for multiplexeddeterminations, where the compounds can be linked to binding compoundsfor detection of reciprocal binding compounds in a sample. The methodsare distinguished by having a plurality of binding events in a singlevessel using a mixture of differentially eTag reporter conjugatedbinding compounds, the release of identifying eTag reporter of thosebinding compounds bound to their target compounds in the same vessel,and the detection of the released identifying tags by separation of thetags in a single run. The eTag reporter are distinguished by having oneor more physical characteristics that allow them to be separated anddetected.

[0051] The method employs a mixture of binding compounds bound to eTagreporters, where each eTag reporter has a characteristic that allows itto be uniquely detected in a single separation run. The method involvescombining the eTag reporter conjugated binding compound with a sample todetermine the presence of a plurality of targets under conditions wherethe binding compounds bind to any reciprocal binding partners to form abinding complex. After sufficient time for binding to occur, the eTagreporters can be released from binding complexes in the same vessel.Various techniques are employed depending upon the nature of the bindingcompounds for releasing the eTag reporters bound to the complex. Thereleased eTag reporters are then separated and identified by theirdifferentiable characteristics free of interference from the eTagreporters still bound to the binding compound. The techniques fordifferentiating between eTag reporters bound to a complex and not boundto a complex, include enzymatic reactions that require the complex toexist for cleavage to occur, modification by using ligand/receptorbinding, where the ligand is part of the binding compound, so that aftercleavage, eTag reporter still bound to the binding compound is modified,dual binding to the target resulting in release of the eTag reporter,where optionally eTag reporter bound to the binding compound ismodified, and the like.

[0052] One set of eTag reporter are distinguished by differences, whichinclude mass as a characteristic. These eTag reporters do not rely ondifferentiation based on oligonucleotides of 2 or more, usually 3 ormore nucleotides, but rather on organic chemical building blocks thatare conveniently combined together to provide for large numbers ofdifferentiable compounds. Therefore, while the original eTag reporter oreTag reporter conjugated to the binding compound can have 2 or morenucleotides, when released from the binding compound, the released eTagreporter will have not more than 3, usually not more than 2 nucleotides.Of particular interest are eTag reporter that are characterized bydifferences in their mass/charge ratio. These compounds aredistinguished by having differences in mobility and are characterized byhaving regions, which serve as (1) a cleavable linking region; (2) amass-modifying region; (3) a charge-modifying region: and (4) adetectable region, where the regions may be separate and distinct orcombined, there being at least two distinct regions that provide for thedifferentiation. These eTag reporters may be combined in kits and assayswith compounds having all of the regions within a single region tofurther expand the number of different compounds used as eTag reportersin a multiplexed determination. These compounds find use with othercompounds where the different regions are present in the same moiety,for example one to two regions, where the charge-modifying region mayalso be the detectable region or the mass-modifying region. By having aplurality of compounds that can serve as identifying molecules, mixturesof target compounds can be assayed in a single vessel. By usingprotocols that result in the release of eTag™ reporters from the bindingcompound that are identifiable due to differences in mobility, theanalysis is greatly simplified, since the eTag reporters will besubstantially free of interfering materials and their differences inmobility will allow for accurate detection and quantitation.

[0053] The eTag reporters will vary depending upon the method ofdetection. Groups of at least 10 eTag reporters bound to 10 differentbinding compounds will be used in the determinations. The eTag reporterswill be characterized by being cleavable from the binding compound inthe same vessel by the same cleavage mechanism, having a sharedcharacteristic that permits separation and individual detection, beingcompatible with the determination method and being in the molecularweight range of about 30 to 3000 dal, usually in the molecular weightrange of about 35 to 1500 dal. The variation may be mass using a massspectrometer, where a magnetic field is used for separation, mass/chargeratio using electrokinesis, where an electric field is used forseparation, which may also include sieving and/or adsorbing polymers,adsorption, using chromatography, e.g gas chromatography, high pressureliquid chromatography, where polar and van der Waal interactions areused for separation, etc.

[0054] For those eTag reporters that rely on mass as a characteristic,the mass unit difference in each eTag reporter when using massspectrometry for analysis need only be one, preferably at least about 2.For electrophoresis, one will usually have at least a 3, usually 5 unitdifference as to the mass/charge ratio, preferably at least about 7, andif one wishes to use shorter distances for separation, 10 or more. Theseunit differences are intended for molecules of similar structure, for aswill be discussed subsequently, structures can affect the mobilitywithout changing the mass/charge ratio.

[0055] For the most part, the eTag reporters that have independentregions will have the following formula:

*ML*C*(D)_(n)

[0056] wherein:

[0057] L is a terminal linking region;

[0058] M is the mass-modifying region;

[0059] C is the charge-modifying region;

[0060] D is the detectable region, being present when the eTag reporteris detected using spectrophotometric measurement and is not present whenthe eTag reporter is detected using mass spectrometric measurement;

[0061] n is 0 or 1, being 1 for spectrophotometric measurement and 0 formass spectrometric measurement; and

[0062] the * intends that M, C and D can be bonded to any of the othergroups at any site, and

[0063] when not independent and distinct regions,

[0064] any of M, C and D may be merged together to provide multiplefunctions in a single region and the regions may be bonded directly toeach other or interspersed with linking groups or regions. That is,parts of one region may be separated by the whole or parts of anotherregion. Also, as indicated earlier, the different regions will be freeof regions comprising oligonucleotides of 3 or more nucleotides, usuallyfree of regions comprising oligonucleotides of 2 or more nucleotides.

[0065] Where the eTag reporter is bound to the binding compound, theeTag reporter will have the following formula:

*MB-L′*C*(D)_(n)

[0066] wherein:

[0067] B is the binding compound bonded to L′;

[0068] L′ is a modified linking group as a result of the bonding to B;and

[0069] the remaining symbols are as defined previously.

[0070] The released eTag reporters will have the following formula:

*ML″*C*(D)_(n)

[0071] wherein:

[0072] L″ is the residue of the linking region, which may include moreor less than the original linking group, by including a portion of thebinding compound or retaining only a portion of the linking region, bycleaving at other than the bond made by joining the linking region andthe binding compound; and

[0073] the remaining symbols are as defined previously.

[0074] Each of the regions may be joined in a variety of ways usingdifferent functionalities and synthetic protocols, where the manner oflinking may serve as one of the regions, for example, having phosphatelinks that result in negatively charged links.

[0075] The linking region functions as the link between the remainder ofthe eTag reporter and the binding compound. L has three aspects: areactive functionality, either inherently or made so by reacting with anactivating moiety; a cleavable linkage, which may be the linkage formedby joining to the binding compound, and a group(s) for joining to one ormore of the other regions. For bonding to the binding compound,different reactive functionalities may be used, depending upon thenature of the binding compound.

[0076] Where the binding compound is an oligonucleotide, that is DNA,RNA, combinations thereof and analogs thereof, e.g. thio analogs, groupsthat react with alcohols will ordinarily be used. Reactive groupsinclude phosphoramidites, e.g. dialkyl phosphoramidites, wherein alkylis of from 1-6 carbon atoms; alkyl, cyanoethyl phosphoramidites, whereinalkyl is of from 1-6 carbon atoms, etc.; trialkyl phosphites orphosphates, where alkyl is of from 1-6 carbon atoms; carboxylic acids orderivatives thereof, such as acyl halides, anhydrides and active esters,e.g. dinitrophenyl ester; active halides, such as α-halomethyloxo-andnon-oxo, where the halo will be of atomic number 17-53, chloro, bromoand iodo; and the like. The products will be esters, both inorganic andorganic acid esters, and ethers. Alternatively, in some cases, one mayuse other than phosphate derivatives as the linking unit, using aminoacids instead, such as glycine and substituted glycines. In thisinstance, the units of the eTag reporter would use analogous chemistryto synthesize the eTag reporter in situ. The exemplary linkers are onlyillustrative and not intended to be exhaustive.

[0077] For the most part for oligonucleotides, cleavage will be at aphosphate bond between two nucleosides cleaved by an enzyme havingnuclease activity e.g. 5′-3′ nuclease activity. Therefore, the linkingregion will usually include a phosphoric acid derivative for coupling tothe terminal hydroxy of an oligonucleotide having an appropriate base,such as adenine, cytosine, guanosine, thymidine and uracil. As will bediscussed subsequently other available hydroxyl groups of the sugar,ribose or deoxyribose, may be substituted with one of the other regions.Where other methods than nuclease activity are used for release of theeTag reporter, then any of the other functionalities may be used forlinking to the oligonucleotide. The linking region will then include afunctional entity that allows for specific cleavage.

[0078] One need not use oligonucleotides for detection of specificnucleic acid sequences. By employing binding compounds that recognize aparticular sequence, either as ssDNA or dsDNA, one may attach adifferent eTag reporter to each of the different binding compounds.Combining the nucleic acid sample with the eTag reporter labeled bindingcompounds results in the binding of the binding compounds to sequencesthat are present in the sample. Various protocols can be used dependingon the nature of the binding compound. For example, oligomers ofheterocyclic compounds, particularly azole compounds, e.g. pyrrole,imidazole, hydroxyimidazole, joined by two atom chains, particularlyhaving —NH— groups, and amino acids, e.g. glycine, alanine, β-alanine,γ-aminobutyric acid, etc. are employed. The azoles are normallyconnected by a two atom bridge containing an —NH— group, desirably fromthe 2 to the 4 or 5 position. These compounds form hairpins that bind inthe minor groove of dsDNA with high affinity and specificity for thesequence. See, for example, U.S. Pat. Nos. 6,090,947 and 5,998,140,which are specifically incorporated by reference herein for thedisclosure of binding sequences.

[0079] By adding the appropriate oligomers to a dsDNA sample, which mayinclude intact or fragmented dsDNA, sequestering the bound oligomersfrom unbound oligomers and releasing the eTag reporters bound to thedsDNA, one can rapidly determine the presence of dsDNA sequences in thesample. Sequestering can be achieved with proteins that bind dsDNA, byhaving ligands bound to the dsDNA, e.g. using PCR with primers carryinga ligand, etc. Alternatively, by having a biotin or other ligand bondedto the eTag reporter conjugated to the binding compound that is retainedwith the binding compound on release of the eTag reporter, one can addthe ligand receptor having a charge opposite to the released eTagreporter, so that in electrophoresis the eTag reporter would migrate inthe opposite direction. The methods can find particular use where thesensitivity of the system is adequate to avoid amplification anddirectly determine the presence of a sequence without denaturation. Thisapproach can find use with detecting infectious organisms, e.g.bacteria, viruses and protista, identifying specific chiasmas,identifying genomes, and the like.

[0080] There are a large number of different functional entities thatare stable under the conditions used for the binding event with thebinding compound and may then be cleaved without affecting adversely theeTag reporter. Functional entities may be cleaved by chemical orphysical methods, involving oxidation, reduction, solvolysis, e.g.hydrolysis, photolysis, thermolysis, electrolysis, chemicalsubstitution, etc. Specific functional entities include thio ethers thatmay be cleaved with singlet oxygen, disulfide that may be cleaved with athiol, diketones that may be cleaved by permanganate or osmiumtetroxide, β-sulfones, tetralkylammonium, trialkylsulfonium,tetralkylphosphonium, etc., where the α-carbon is activated withcarbonyl, nitro, etc., that may be cleaved with base, quinones whereelimination occurs with reduction, substituted benzyl ethers that can becleaved photolytically, carbonates that can be cleaved thermally, metalchelates, where the ligands can be displaced with a higher affinityligand, as well as many other functional entities that are known in theliterature. Cleavage protocols are described in U.S. Pat. Nos.5,789,172, 6,001,579, and references cited therein.

[0081] The eTag reporters find use in determinations involving aplurality of target entities. Usually, one will be interested in atleast about 3 target entities, more usually at least 5, frequently atleast about 10 or more, and may be interested in at least about 20 ormore, even about 100 or more. The number of eTag reporters will usuallybe equal to the number of target entities, although in some situations,the same eTag reporter may be used to identify a plurality of relatedtarget entities and one may then deconvolute the results as toindividual target entities. The eTag reporters bound to the bindingmembers can be added individually or in combination to the sample andthen processed to determine the presence of the target entities.

[0082] Of interest is to have two eTag reporters that are closelysimilar in mobility, usually closer in mobility to each other than tounrelated eTag reporters. Where there are paired situations to beanalyzed, such as alleles, MHC antigens, single nucleotidepolymorphisms, etc., by having the eTag reporters in proximity in theelectropherogram, particularly where they have distinguishabledetectable regions, e.g. fluorescers fluorescing at differentwavelengths, one obtains a quick determination if none, one or both ofthe pairs are present in the sample.

[0083] Genetic analyses may take many forms and involve determinationsof different information. Genetic analyses are involved with sequencing,detection of specific sequences as related to the presence of specificgenes or regulatory sequences, identification of organisms,identification of transcription events as related to different cells,different cell stages and external stimuli, identification of singlenucleotide polymorphisms, alleles, repetitive sequences, plastid DNA,mitochondrial DNA, etc., forensic medicine, and the like. In each caseone has a complex sample to be assayed, where one is interested innumerous binding events. By providing for a unique eTag reporter foreach event, one can perform simultaneously a number of assays in thesame flask and with a single sample or a few aliquots of the sample. Forexample, where an assay involves a single nucleotide in each vessel, onewould use four vessels, one for each nucleotide. In most cases, the eTagreporters can be separated from other components of the assay mixture tosubstantially reduce interference from these other components whenassaying for the eTag reporters.

[0084] There are a number of genetic analyses that involve cleavage of aphosphate bond of a nucleic acid sequence as a result of hybridization.For the most part, the initial step will be in solution, although onemay have one or more reagents bound to a solid support in the first andsucceeding stages of the determination. One technique is described inU.S. Pat. Nos. 5,876,930 and 5,723,591, where a primer and a probe arebound to a target sequence and by extending the primer with a DNApolymerase having 5′-3′ nuclease activity, the terminal nucleotides arecleaved as the polymerase processes along the target DNA. By having aneTag reporter bonded to the terminal and/or internal nucleotide(s), theeTag reporter will be released when the target nucleic acid is present.Another technique employs an enzyme referred to as a cleavase, whichrecognizes a three member complex of the target nucleic acid, a primerand a probe. See, U.S. Pat. No. 5,719,028. Attached to the terminus ofthe probe is an eTag reporter that is released by the cleavase, wherethe three membered complex is formed.

[0085] For detecting single nucleotide polymorphisms (“snps”), varioustechniques can be employed of varying complexity. In one technique, aprimer is employed that terminates at the nucleotide immediatelypreceding the snp. One can have the eTag reporter bound to the primerand a ligand bound to the nucleotide reciprocal to the snp. One caneither have 4 vessels, each with a different labeled nucleotide or onevessel with each of the labeled nucleotides having a different label.Various polymerases having 3′-5′ editing can be used to ensure thatmismatches are rare. The extended primers may then be captured, forexample, by having a ligand, e.g. biotin, and contacting the extensionmixture with the reciprocal receptor, e.g. streptavidin, bound to asupport and the eTag reporter released and analyzed. By grouping targetsof interest having the same nucleotide for the snp, the assay may bemultiplexed for a plurality of targets. Other techniques include havingprobes where the snp is mismatched. The mismatching nucleotide islabeled with the eTag reporter. When the snp is present, the eTagreporter labeled nucleotide will be released for detection. See U.S.Pat. No. 5,811,239.

[0086] In another variation, one may ligate a primer and a probe, whereone is 3′ of the other when hybridized to a target nucleic acid. Byhaving one of the pair of primer and probe with an eTag reporter with acleavable linkage and the other of the pair with an agent capable ofcausing cleavage of the cleavable linkage in conjunction with anotheragent, the primer and probe may be ligated together when bound to thetarget. One can release the ligated pair from the target, e.g. heat, andrecycle by cooling the mixture to allow for hybridization of the primerand probe, ligating primer and probe bound to target and then denaturingto release the ligated primer and probe, amplifying the number ofligated primers and probes. Once the desired degree of amplification hasbeen achieved, one may provide the additional reagent resulting inrelease of the eTag reporters.

[0087] Where PCR or other amplification reaction is used involving aprimer, the primer can be labeled with a ligand that allows forsequestering of the amplified DNA, one can then sequester the DNA bymeans of a receptor reciprocal to the ligand, which receptor is bound toa support and add probes labeled with eTag reporters specific for theprobe sequence. After hybridization and washing to removenon-specifically bound and unbound nucleic acid, the eTag reporters arereleased and analyzed.

[0088] Instead of nucleic acid assays, one may be interested in proteinassays. For determining a mixture of proteins, one may use intact cells,intact viruses, viral infected cells, lysates, plastids, mitochondria orother organelles, fractionated samples, or other aggregation ofproteins, by themselves or in conjunction with other compounds. Anysource of a mixture of proteins can be used, where there is an interestin identifying a plurality of proteins.

[0089] Proteomics has come to the fore, where one is interested incellular expression during metabolism, mitosis, meiosis, in response toan external stimulus, e.g. drug, virus, change in physical or chemicalcondition, involving excess or deficient nutrients and cofactors,stress, aging, presence of particular strains of an organism andidentifying the organism and strain, multiple drug resistance, and thelike. It is necessary to have a means for identifying a large number ofproteins in a single sample, as well as providing some quantitation ofthe different proteins being detected. In one assay one may use bindingproteins specific for the target proteins. One group of binding proteinsis bound to a support, such as a vessel or channel wall, particles,magnetic or non-magnetic, e.g. latex particles, dextrose, sepharose,cellulose, etc., where the support permits sequestering the targetproteins to the support. Most commonly, antibodies, particularlymonoclonal antibodies rather than antisera, will be used, although thelatter may also find use. In some situations other receptors may finduse, such as lectins, enzymes, surface membrane proteins, etc. and insome situations, ligands for the proteins may be employed. Thereciprocal-binding members, receptors and ligands, may be bound to thesupport through covalent or non-covalent bonding. Activated surfacesfind use, where the surface has an active functional group that willreact with the reciprocal-binding member to provide for stable bindingto the surface, e.g. silyl chloride modified glass, cyanogen bromidemodified polysaccharides, etc. Proteins bind tightly to some plasticsurfaces, so that no covalent bonding is required. Ligands have or canbe provided with active functional groups for bonding to the surface. Ifdesired the binding to the surface can be accomplished in two steps bybonding a ligand to the reciprocal binding member and binding a ligandbinding member to the support, for example, biotin as the ligand andstrept/avidin as the ligand binding member, or one may have anti-Igbound to the surface to bind to antibodies bound to the target protein.In addition, where a change in environment is localized, one may have alarge concentration of a counteracting agent, e.g. a large amount ofbuffer at pH 7, for example, ≧200 mM phosphate, where ammonia isproduced that creates a localized basic environment.

[0090] The sample is combined with the reciprocal binding member, whichmay be bound to the support or subsequently bound to the support. Afterwashing away the other components of the mixture, receptor for thetarget protein labeled with eTag reporter molecules specific for theparticular receptor are added to the bound target protein, so as tobecome bound to the support through the target protein. One or more eTagreporter molecules will be bound to the receptor, usually not more thanabout 20, frequently not more than about 10. The number will be limitedby the degree of loss of the binding affinity as the number of eTagreporter molecules is increased. Normally, the support bound receptorand the eTag reporter labeled receptor will bind to different epitopesof the target protein, although in some situations where the target hasa plurality of the same epitope, the receptors may be specific for thesame epitope. After washing away all eTag reporter labeled receptor thatis not specifically bound to the target protein(s), the eTag reportermolecules are released and assayed.

[0091] Where the target permits binding of two reciprocal bindingmembers or where an additional reagent is provided which permits thisevent, one can use determinations involving “channeling” or energytransfer. See, for example, U.S. Pat. Nos. 5,843,666 and 5,573,906.There are numerous methodologies involving channeling in the literature,where for the most part, the channeling was involved in producing adirectly detectable signal, usually a change in absorption or emissionof light. Channeling involves having two reagents, where the firstreagent, when in proximity to the second reagent, produces a detectablesignal. For the eTag reporter, the detectable signal is the release ofthe eTag reporter from the binding component. The release will usuallybe a function of the production of a short-lived entity, such as achemical species or a photoactivated excited species, but may be theresult of changing the local environment as compared to the bulksolution. So far as the chemical species, illustrative species includesinglet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals. Twoentities are employed that have reciprocal binding members that bind tothe same target moiety. One of the entities generates an active species.The other entity has a susceptible functionality that interacts with theactive species resulting in release of the eTag reporter or responds tothe changed local environment to release the eTag reporter. Either theactive species is short lived, so that it will not create significantbackground because beyond its vicinity, the active species becomesinactive or a scavenger is employed that efficiently scavenges theactive species, so that it is not available to react with thesusceptible functionality that is not bound to the target.

[0092] Generators of reactive species include enzymes, such as oxidases,such as glucose oxidase, xanthene oxidase, D-amino acid oxidase,NADH-FMN oxidoreductase, galactose oxidase, glyceryl phosphate oxidase,sarcosine oxidase, choline oxidase and alcohol oxidase, that producehydrogen peroxide, horse radish peroxidase, that produces hydroxylradical, various dehydrogenases that produce NADH or NADPH, urease thatproduces ammonia to create a high local pH. One cleavable link can bebased on the oxidation of sulfur or selenium, where a thioether,sulfoxide, or selenium analog thereof, is present at the α- orβ-position in relation to an activating group, which makes the hydrogenat to the activating group acidic and capable of being removed by base,so as to release the oxidized functionality to which is attached theeTag reporter or to be subject to oxidation with release of the eTagreporter. Alternatively, one may use metal chelates that are stable atone oxidation state and unstable at another oxidation state. Othercompounds include a α-substituted methylquinones, which have an eTagreporter bonded through a leaving group, such as sulfonyl, oxy, amino,etc.

[0093] By using a heterogeneous system, a first agent for causingcleavage may be bound to a surface to provide an environment for releaseof the eTag reporter when bound to the surface. Where a second agent isrequired to cause the release of the eTag reporter, the second agent isadded after sufficient time for the eTag reporter conjugated bindingcompound to become bound to the surface. Where the target is a nucleicacid, the nucleic acid may be bound to the first agent containingsurface by having ssDNA binding proteins bound to the surface or otherconvenient means known in the art. Once the target is bound to thesurface, the eTag reporter conjugated oligonucleotides homologous thetarget nucleic acid sequences are added, followed by the second agent.With ligands and proteins, one can have receptors, which bind at onesite, on the surface and eTag reporter binding compounds that bind at adifferent site forming what is referred to in the art as a “sandwich.”

[0094] For singlet oxygen, one may use various sensitizers, such assquarate derivatives. See, for example, Ullman, et al., Proc. Natl.Acad. Sci. USA 91,5426-5430(1994). Examples of combinations that finduse in this invention may be found in U.S. Pat. Nos. 5,536,498;5,536,834; references cited therein; H. H. Wasserman and R. W. Murray.Singlet Oxygen. Academic Press, New York (1979); A. L. Baumstark,Singlet Oxygen, Vol. 2, CRC Press Inc., Boca Raton, Fla. 1983. Othercleavage mechanisms may be found in WO99/64519; WO99/13108; WO98/01533and WO97/28275.

[0095] Singlet oxygen reacts with a wide variety of double bonds, withcleavage of the double bond to an oxo group with separation of the eTagreporter. Illustrative olefins include vinyl sulfides, vinyl ethers,enamines, imines substituted at the carbon atoms with an α-methine (CH,a carbon atom having at least one hydrogen atom), where the vinyl groupmay be in a ring, the heteroatom may be in a ring, or substituted on thecyclic olefinic carbon atom, and there will be at least one and up tofour heteroatoms bonded to the olefinic carbon atoms. The resultingdioxetane may decompose spontaneously, by heating above ambienttemperature, usually below about 75° C., reaction with acid or base, orphotolytically in the absence or presence of a sensitizer. Numerousarticles describe a variety of compounds that can be decomposed withsinglet oxygen, where the articles are frequently interested in lightemission, so that the compounds have more complicated structures thanare required for the subject purposes, where only cleavage is requiredfor release of the eTag reporter from the binding compound. Therefore,for the most part, synthetic convenience, stability under the conditionsof the linking to the binding compound and conditions of the binding,and efficiency of release will be the primary factors in selecting aparticular structure.

[0096] Articles of interest which are illustrative of a much largerliterature include: Adam and Liu, J. Amer. Chem. Soc. 94, 1206-1209,1972, Ando, et al., J.C.S. Chem. Comm. 1972, 477-8, Ando, et al.,Tetrahedron 29, 1507-13, 1973, Ando, et al., J. Amer. Chem. Soc. 96,6766-8, 1974, Ando and Migita, ibid 97, 5028-9, 1975, Wasserman andTerao, Tetra. Lett. 21, 1735-38, 1975, Ando and Watanabe, ibid 47,4127-30, 1975, Zaklika, et al., Photochemistsry and Photobiology 30,35-44, 1979, and Adam, et al., Tetra. Lett. 36, 7853-4, 1995. See also,U.S. Pat. No. 5,756,726.

[0097] The formation of dioxetanes is obtained by the reaction ofsinglet oxygen with an activated olefin substituted with an eTagreporter at one carbon atom and the binding compound at the other carbonatom of the olefin. See, for example, U.S. Pat. No. 5,807,675. Thesecompounds may be depicted by the following formula:

(eTag reporter−W)(X)_(n)C_(α)═C_(β)(Y)(Z)

[0098] wherein:

[0099] W may be a bond, a heteroatom, e.g. O, S, N, P, M (intending ametal that forms a stable covalent bond), or a functionality, such ascarbonyl, imino, etc., and may be bonded to X or C_(α);

[0100] at least one X will be aliphatic, aromatic, alicyclic orheterocyclic and bonded to C_(α) through a hetero atom, e.g. N, O, or Sand the other X may be the same or different and may in addition behydrogen, aliphatic, aromatic, alicyclic or heterocyclic, usually beingaromatic or aromatic heterocyclic wherein one X may be taken togetherwith Y to form a ring, usually a heterocyclic ring, with the carbonatoms to which they are attached, generally when other than hydrogenbeing from about 1 to 20, usually 1 to 12, more usually 1 to 8 carbonatoms and one X will have 0 to 6, usually 0 to 4 heteroatoms, while theother X will have at least one heteroatom and up to 6 heteroatoms,usually 1 to 4 heteroatoms;

[0101] Y will come within the definition of X, usually being bonded toC_(β) through a heteroatom and as indicated may be taken together with Xto form a heterocyclic ring;

[0102] Z will usually be aromatic, including heterocyclic aromatic, offrom about 4 to 12, usually 4 to 10 carbon atoms and 0 to 4 heteroatoms,as described above, being bonded directly to C_(β) or through aheteroatom, as described above;

[0103] n is 1 or 2, depending upon whether the eTag reporter is bondedto C_(α) or X;

[0104] wherein one of Y and Z will have a functionality for binding tothe binding member or be bound to the binding member.

[0105] While not depicted in the formula, one may have a plurality ofeTag reporters in a single molecule, by having one or more eTagreporters joined to one or both Xs.

[0106] Illustrative compounds include S-(eTag reporter)3-thiolacrylicacid, N-(eTag reporter), N-methyl4-amino-4-butenoic acid, O-(eTagreporter), 3-hydroxyacrolein, N-(4-carboxyphenyl)2-(eTag reporter)imidazole, oxazole, and thiazole.

[0107] Also of interest are N-alkyl acridinyl derivatives, substitutedat the 9 position with a divalent group of the formula:

—(CO)X¹(A)-

[0108] wherein:

[0109] X¹ is a heteroatom selected from the group consisting of O, S, N,and Se, usually one of the, first three; and

[0110] A is a chain of at least 2 carbon atoms and usually not more than6 carbon atoms substituted with an eTag reporter, where preferably theother valences of A are satisfied by hydrogen, although the chain may besubstituted with other groups, such as alkyl, aryl, heterocyclic, etc.groups, A generally being not more than 10 carbon atoms.

[0111] Also of interest are heterocyclic compounds, such asdiheterocyclopentadienes, as exemplified by substituted imidazoles,thiazoles, oxazoles, etc., where the rings will usually be substitutedwith at least one aromatic group and in some instances hydrolysis willbe necessary to release the eTag reporter.

[0112] Also of interest are tellurium (Te) derivatives, where the Te isbonded to an ethylene group having a hydrogen atom β to the Te atom,wherein the ethylene group is part of an alicyclic or heterocyclic ring,that may have an oxo group, preferably fused to an aromatic ring and theother valence of the Te is bonded to the eTag reporter. The rings may becoumarin, benzoxazine, tetralin, etc.

[0113] The mass-modifying region, when not including thecharge-modifying region or the detectable label, will usually be aneutral organic group, aliphatic, alicyclic, aromatic or heterocyclic,where the heteroatoms will be neutral under the conditions employed forthe assay protocol. The heteroatoms may be oxygen as oxy or non-oxo- oroxo-carbonyl, sulfur as thio or thiono, halo, nitrogen as amide, nitroor cyano, phosphorous as phosphite or phosphate triester, etc.Conveniently, the region may be methylene, including polymethyene,alkyleneoxy, including polyalkyleneoxy, particularly alkylene of 2-3carbon atoms, aryl or substituted aryl, such as phenylene, diphenylene,cyanophenylene, nitrophenylene, thiophenylene, chlorophenylene,furanylene, amino acids, such as N-acyl glycinamide and polyglycinamide,including substituted glycinamides, cyclopentylene, bis-biphenylene-E,where E is carbonyl, oxy, thio, ureido, methylene, isopropylene, and thelike; etc. The mass-modifying region will generally be from about 1 to100, more usually 1 to 60 atoms other than hydrogen, generally having atleast one carbon atom and up to 60 carbon atoms and from about 0 to 40heteroatoms, usually about 0 to 30 heteroatoms.

[0114] The charge-modifying region will vary depending upon the othergroups present and whether one wishes to reduce the number ofunneutralized charges in the molecule or increase the number ofunneutralized charges. Charges in the molecule may come from other thanthe charge-modifying group, such as the label, connecting groups betweenregions may be included in the charge modifying region, the linkingregion, and any residue of the binding compound that is retained withthe eTag reporter. For the most part, the eTag reporter will have anoverall negative charge, although in some instances, there may be anoverall positive charge, particularly if positive and negative eTagreporters are to be determined in the same electrophoretic separation.Negative charges can be provided by phosphate, including phosphonate,phosphinate, thiophosphate, etc., borate, carboxylate, sulfonate,enolate, phenoxide, etc. Positive charges can be provided by amines andsubstituted amines, e.g. ammonium, sulfonium, hydrazine, imine, amidine,metal ions, particularly as chelates and metallocenes, etc. Thecharge-modifying region may have from 1 to 60 atoms other than hydrogen,usually from about 1 to 30 atoms, where there will be at least oneheteroatom, which may be oxygen, nitrogen, sulfur, boron, phosphorous,metal ion, etc.

[0115] One may combine the mass-modifying and charge-modifying functionsin a single region in a convenient manner using poly(amino acids), wherethe naturally occurring aspartate and glutamate may serve to providenegative charges, and the naturally occurring lysine, arginine andhistidine may serve to provide positive charges. However, one may wishto use unnatural amino acids, such as sulfonic, phosphonic, and boronicacid substituted amino acids. By appropriate choice in conjunction withthe other regions, a large number of different mobilities can beachieved. When used in combination with mass-modifying regions, thenumber of eTag reporters having different mobilities is greatlyexpanded.

[0116] One may use combinations of substituted diols or diamines anddibasic acids, where the substituents are charged, to form diesters anddiamides. Illustrative of such oligomers are the combination of diols ordiamino, such as 2,3-dihydroxypropionic acid, 2,3-dihydroxysuccinicacid, 2,3-diaminosuccinic acid, 2,4-dihydroxyglutaric acid, etc. Thediols or diamino compounds can be linked by dibasic acids, which dibasicacids include the inorganic dibasic acids indicated above, as well asdibasic acids, such as oxalic acid, malonic acid, succinic acid, maleicacid, furmaric acid, carbonic acid, citric acid, tartaric acid, etc.Alternatively, one may link the hydroxyls or amines with alkylene orarylene groups, dicarbonyls, activated dihalo compounds, etc. Othercombinations include substituted dithiols, that can be copolymerizedwith dienes, activated dihalo compounds, etc. Thus, by appropriateselection of the different monomers, low order oligomers can be producedthat may then be separated by molecular weight.

[0117] The detection region may include any label that can be detectedspectrophotometrically and/or electrochemically. A wide variety oflabels are available for detection in an electrophoretic device.Commonly used fluorescers include, fluorescein and fluoresceinderivatives, lanthanide dyes, rhodamine and rhodamine derivatives, Cy-5,Cy-3, HEX, TET, squarates, and cyanine dyes. The dyes may be charged oruncharged, so as to add or diminish the overall charge of the molecule.Electrochemical labels also find use, such as ferrocene and rutheniumcomplexes.

[0118] For economic and operational reasons, it is generally desirableto use as few lasers for excitation as feasible. Therefore, it will bedesirable to use combinations of energy absorbers/transmitters,frequently a fluorescer, and energy receivers/emitters, usually afluorescer, keeping the energy absorber constant for excitation whereenergy exchange between the two entities allows for variation in theemission wavelength due to changes in the Stokes shift. Combinations ofdyes include fluorescein and HEX, (ex_(488 nm), em_(560 nm)), andphthalocyanine (ex_(488 nm), em_(690 nm)). One can provide for variouscombinations of fluorescers to be bound in proper proximity for energytransfer. A ribosyl group in the linking region or the mass-modifyingregion provides for one hydroxyl group for linkage of a member of anenergy transfer pair and two hydroxyls for insertion into the chain,while deoxyribose substituted with two fluorescers can react with anhydroxyl group as a side chain. The particular unit used to which themembers of the energy transfer pair are bonded can be selected toprovide mass-modification and/or charge-modification.

[0119] The mobility of the eTag reporter will not only depend on themass/charge ratio according to the formula (M/z)^(2/3), but will alsodepend on structure. Entities within the eTag reporter that are rigidand extend the molecule enhance the drag and therefore reduce themobility. Therefore by using rigid groups, such as aromatics, 5- and6-membered heterocyclics, e.g. tetrahydrofuran, polyenes andpolyacetylenes, one can enhance differences in mobility even while theratio of mass to charge is not significantly different.

[0120] Synthesis of eTags comprising nucleotides can be easily andeffectively achieved via assembly on a solid phase support during probesynthesis using standard phosphoramidite chemistries. The eTag reportersare assembled at the 5′-end of probes after coupling of a finalnucleosidic residue, which becomes part of the eTag reporter during theassay. One may have a nucleotide triphosphate bonded to one of thetermini of the building blocks of the eTag reporter. In one approach,the eTag reporter is constructed sequentially from a single or severalmonomeric phosphoramidite building blocks (one containing a detectableregion, e.g. dye), which are chosen to generate eTag reporters withunique electrophoretic mobilities based on their mass to charge ratio.The eTag reporter is thus composed of monomeric units of variable chargeto mass ratios bridged by phosphate linkers (FIG. 1A). The separation ofeTag reporters, which differ by 9 mass units (Table 1) has beendemonstrated. The nucleosidic phosphoramidites employed for eTagreporter synthesis are initially either modified or natural residues.Fluorescein has been the initial dye employed but other dyes can be usedas well, as illustrated in FIG. 1A.

[0121] Some of the combinations of phosphoramidite building blocks withtheir predicted elution times are presented in Table 2. As shown in FIG.B, eTag reporters are synthesized to generate a continuous spectrum ofsignals, one eluting after another with none of them coeluting (FIG.1B). TABLE 1 eTag reporters that have been separated on a LabCard (Seeexperimental section for description.)(detection: 4.7 cm; 200 V/cm).E-Tag Elution Time on CE (sec) Mass

385 778

428 925

438 901

462 994

480 985

555 961

[0122] TABLE 2 Predicted and experimental (*) elution times of eTagreporters. C₃, C₆, C₉, C₁₈, are commercially available phosphoramiditespacers from Glen Research, Sterling VA. The units are derivatives ofN,N-diisopropyl, O-cyanoethyl phosphoramidite, which is indicated by“Q”. C₃ is DMT (dimethoxytrityl)oxypropyl Q; C₆ is DMToxyhexyl Q; C₉ isDMToxy(triethyleneoxy) Q; C₁₂ is DMToxydodecyl Q; C₁₈ isDMToxy(hexaethyleneoxy) Q. Etag Charge Elution Time

−9 41.12

−8 43.72

−9 45.66

−8 48.14

−7 51.21

−6 53.53

−6 55.13

−5 57.66

−5 60.00

−5 62.86

−6 65.00*

−5 67.50*

−4 69.61

−4 72.00*

[0123] All of the above eTag reporters work well and are easilyseparable and elute after 40 minutes. To generate eTag reporters thatelute faster, highly charged low molecular weight eTag reporters arerequired. Several types of phosphoramidite monomers allow for thesynthesis of highly charged eTag reporters with early elution times. Useof dicarboxylate phosphoramidites (FIG. 1C), allows for the addition of3 negative charges per coupling of monomer. Polyhydroxylatedphosphoramidites (FIG. 1D) in combination with a common phosphorylationreagent enable the synthesis of highly phosphorylated eTag reporters.Combinations of these reagents with other mass modifier linkerphosphoramidites allow for the synthesis of eTag reporters with earlyelution times.

[0124] The aforementioned label conjugates with differentelectrophoretic mobility permit a multiplexed amplification anddetection of multiple targets, e.g. nucleic acid targets. The labelconjugates are linked to oligonucleotides in a manner similar to thatfor labels in general, by means of linkages that are enzymaticallycleavable. It is, of course, within the purview of the present inventionto prepare any number of label conjugates for performing multiplexeddeterminations. Accordingly, for example, with 40 to 50 different labelconjugates separated in a single separation channel and 96 differentamplification reactions with 96 separation channels on a singlemicrofluidic chip, one can detect 4000 to 5000 single nucleotidepolymorphisms.

[0125] The separation of eTag reporters, which differ by 9 mass units(Table 1) has been demonstrated as shown in FIG. 9. The penultimatecoupling during probe synthesis is initially carried out usingcommercially available modified (and unmodified) phosphoramidites (Table2). This residue is able to form hydrogen bonds to its partner in thetarget strand and is considered a mass modifier but could potentially bea charge modifier as well. The phosphate bridge formed during thiscoupling is the linkage severed during a 5′-nuclease assay. The finalcoupling is done using a phosphoramidite analogue of a dye. Fluoresceinis conveniently employed, but other dyes can be used as well.

[0126] One synthetic approach is outlined in Scheme 1. Starting withcommercially available 6-carboxyfluorescein, the phenolic hydroxylgroups are protected using an anhydride. Isobutyric anhydride inpyridine was employed but other variants are equally suitable. It isimportant to note the significance of choosing an ester functionality asthe protecting group. This species remains intact though thephosphoramidite monomer synthesis as well as during oligonucleotideconstruction. These groups are not removed until the synthesized oligois deprotected using ammonia. After protection, the crude material isthen activated in situ via formation of an N-hydroxy succinimide ester(NHS-ester) using DCC as a coupling agent. The DCU byproduct is filteredaway and an amino alcohol is added. Many amino alcohols are commerciallyavailable some of which are derived from reduction of amino acids. Onlythe amine is reactive enough to displace N-hydroxy succinimide.

[0127] Upon standard extractive workup, a 95% yield of product isobtained. This material is phosphitylated to generate thephosphoramidite monomer (Scheme 1). For the synthesis of additional eTagreporters, a symmetrical bisamino alcohol linker is used as the aminoalcohol (Scheme 2). As such the second amine is then coupled with amultitude of carboxylic acid derivatives (Table 1) prior to thephosphitylation reaction. Using this methodology hundreds even thousandsof eTag reporters with varying charge to mass ratios can easily beassembled during probe synthesis on a DNA synthesizer using standardchemistries.

[0128] Additional eTag reporters are accessed via an alternativestrategy which uses 5-aminofluorescein as starting material (Scheme 3).Addition of 5-aminofluorescein to a great excess of a diacid dichloridein a large volume of solvent allows for the predominant formation of themonoacylated product over dimer formation. The phenolic groups are notreactive under these conditions. Aqueous workup converts the terminalacid chloride to a carboxylic acid. This product is analogous to6-carboxy fluorescein and using the same series of steps is converted toits protected phosphoramidite monomer (Scheme 3). There are manycommercially available diacid dichorides and diacids, which can beconverted to diacid chlorides using SOCl₂ or acetyl chloride. Thismethodology is highly attractive in that a second mass modifier is used.As such, if one has access to 10 commercial modified phosphoramiditesand 10 diacid dichlorides and 10 amino alcohols there is a potential for1000 different eTag reporters. There are many commercial diaciddichlorides and amino alcohols (Table 3). These synthetic approaches areideally suited for combinatorial chemistry. TABLE 3 Mass and chargemodifiers that can be used for conversion of amino dyes into eTagreporter phosphoramidite monomers.

[0129]

[0130] Substituted aryl groups can serve as both mass- andcharge-modifying regions. (Table 4) Various functionalities may besubstituted onto the aromatic group, e.g. phenyl, to provide mass aswell as charges to the eTag reporter. The aryl group may be a terminalgroup, where only one linking functionality is required, so that a freehydroxyl group may be acylated, may be attached as a side chain to anhydroxyl present on the eTag reporter chain, or may have twofunctionalities, e.g. phenolic hydroxyls, that may serve for phophiteester formation and othe substitutients, such as halo, haloalkyl, nitro,cyano, alkoxycarbonyl, alkylthio, etc. where the groups may be chargedor uncharged. TABLE 4 Benzoic acid derivatives as mass and chargemodifiers. (Mass is written below each modifier)

get,0051 122 138

151 176

181 191

198 214

226

249

258 309

[0131] A variety of maleimide derivatized eTag reporters have also beensynthesized. These compounds were subsequently bioconjugated to 5′-thioladorned DNA sequences and subjected to the 5′-nuclease assay. Thespecies formed upon cleavage are depicted in Table 5. TABLE 5 eTagreporters derived from maleimide-linked precursors.

1

2

3

4

5

6

7

8

9

10

[0132] The eTag reporter may be assembled having an appropriatefunctionality at one end for linking to the binding compound. Thus foroligonucleotides, one would have a phosphoramidite or phosphate ester atthe linking site to bond to an oligonucleotide chain, either 5′ or 3′,particularly after the oligonucleotide has been synthesized, while stillon a solid support and before the blocking groups have been removed.While other techniques exist for linking the oligonucleotide to the eTagreporter, such as having a functionality at the oligonucleotide terminusthat specifically reacts with a functionality on the eTag reporter, suchas maleimide and thiol, or amino and carboxy, or amino and keto underreductive amination conditions, the phosphoramidite addition ispreferred. For a peptide-binding compound, a variety of functionalitiescan be employed, much as with the oligonucleotide functionality,although phosphoramidite chemistry may only occasionally be appropriate.Thus, the functionalities normally present in a peptide, such ascarboxy, amino, hydroxy and thiol may be the targets of a reactivefunctionality for forming a covalent bond.

[0133] Of particular interest in preparing eTag reporter labeled nucleicacid binding compounds is using the solid support phosphoramiditechemistry to build the eTag reporter as part of the oligonucleotidesynthesis. Using this procedure, one attaches the next succeedingphosphate at the 5′ or 3′ position, usually the 5′ position of theoligonucleotide chain. The added phosphoramidite may have a naturalnucleotide or an unnatural nucleotide. Instead of phosphoramiditechemistry, one may use other types of linkers, such as thio analogs,amino acid analogs, etc. Also, one may use substituted nucleotides,where the mass-modifying region and/or the charge-modifying region maybe attached to the nucleotide, or a ligand may be attached to thenucleotide. In this way, phosphoramidite links are added comprising theregions of the eTag reporter, whereby when the synthesis of theoligonucleotide chain is completed, one continues the addition of theregions of the eTag reporter to complete the molecule. Conveniently, onewould provide each of the building blocks of the different regions witha phosphoramidite or phosphate ester at one end and a blockedfunctionality, where the free functionality can react with aphosphoramidite, mainly a hydroxyl. By using molecules for the differentregions that have a phosphoramidite at one site and a protected hydroxylat another site, the eTag reporter can be built up until the terminalregion, which does not require the protected hydroxyl.

[0134] Illustrative of the synthesis would be to employ a diol, such asan alkylene diol, polyalkylene diol, with alkylene of from 2 to 3 carbonatoms, alkylene amine or poly(alkylene amine) diol, where the alkylenesare of from 2 to 3 carbon atoms and the nitrogens are substituted, forexample with blocking groups or alkyl groups of from 1-6 carbon atoms,where one diol is blocked with a conventional protecting group, such asa dimethyltrityl group. This group can serve as the mass-modifyingregion and with the amino groups as the charge-modifying region as well.If desired, the mass modifier can be assembled by using building blocksthat are joined through phosphoramidite chemistry. In this way thecharge modifier can be interspersed between the mass modifier. Forexample, one could prepare a series of polyethylene oxide moleculeshaving 1, 2, 3, n units. Where one wished to introduce a number ofnegative charges, one could use a small polyethylene oxide unit andbuild up the mass and charge-modifying region by having a plurality ofthe polyethylene oxide units joined by phosphate units. Alternatively,by employing a large spacer, fewer phosphate groups would be present, sothat without large mass differences, one would have large differences inmass-to-charge ratios.

[0135] The chemistry that is employed is the conventional chemistry usedin oligonucleotide synthesis, where building blocks other thannucleotides are used, but the reaction is the conventionalphosphoramidite chemistry and the blocking group is the conventionaldimethoxyltrityl group. Of course, other chemistries compatible withautomated synthesizers can also be used, but there is no reason to addadditional complexity to the process.

[0136] For the peptides, the eTag reporters will be linked in accordancewith the chemistry of the linking group and the availability offunctionalities on the peptide binding compound. For example, with Fabfragments specific for a target compound, a thiol group will beavailable for using an active olefin, e.g. maleimide, for thioetherformation. Where lysines are available, one may use activated esterscapable of reacting in water, such as nitrophenyl esters orpentafluorophenyl esters, or mixed anhydrides as with carbodiimide andhalf-ester carbonic acid. There is ample chemistry for conjugation inthe literature, so that for each specific situation, there is ampleprecedent in the literature for the conjugation.

[0137] Once the binding compound conjugated with the eTag reporter hasbeen prepared, it may find use in a number of different assays, many ofwhich have already been discussed. The samples may be processed usinglysing, nucleic acid separation from proteins and lipids and vice versa,and enrichment of different fractions. For nucleic acid relateddeterminations, the source of the DNA may be any organism, prokaryoticand eukaryotic cells, tissue, environmental samples, etc. The DNA or RNAmay be isolated by conventional means, RNA may be reverse transcribed,DNA may be amplified, as with PCR, primers may be used with ligands foruse in subsequent processing, the DNA may be fragmented usingrestriction enzymes, specific sequences may be concentrated or removedusing homologous sequences bound to a support, or the like. Proteins maybe isolated using precipitation, extraction, and chromatography. Theproteins may be present as individual proteins or combined in variousaggregations, such as organelles, cells, viruses, etc. Once the targetcomponents have been preliminarily treated, the sample may then becombined with the eTag reporter targeted binding proteins.

[0138] For a nucleic acid sample, after processing, the probe mixture ofeTag reporters for the target sequences will be combined with the sampleunder hybridization conditions, in conjunction with other reagents, asnecessary. Where the reaction is heterogeneous, the target sequence willhave a ligand for binding to a reciprocal binding member forsequestering hybrids to which the eTag reporter is bound. In this case,all of the DNA sample carrying the ligand will be sequestered, both withand without eTag reporter labeled probe. After sequestering the sample,and removing non-specific binding eTag reporter labeled probe under apredetermined stringency based on the probe sequence, using washing atan elevated temperature, salt concentration, organic solvent, etc., theeTag reporter is released into an electrophoretic buffer solution foranalysis.

[0139] For a homogeneous assay, the sample, eTag reporter labeled probemixture and ancillary reagents are combined in a reaction mixturesupporting the cleavage of the linking region. The mixture may beprocessed to separate the eTag reporters from the other components ofthe mixture. The mixture, with or without eTag reporter enrichment, maythen be transferred to an electrophoresis device, usually a microfluidicor capillary electrophoresis device and the medium modified as requiredfor the electrophoretic separation. Where one wishes to remove from theseparation channel intact eTag reporter molecules, a ligand is bound tothe eTag reporter that is not released when the eTag reporter isreleased. Alternatively, by adding a reciprocal binding member that hasthe opposite charge of the eTag reporter, so that the overall charge isopposite to the charge of the eTag reporter, these molecules willmigrate toward the opposite electrode from the released eTag reportermolecules. For example, one could use biotin and streptavidin, wherestreptavidin carries a positive charge. In the case of anoligonucleotide, the eTag reporter label would be bonded to at least twonucleotides, where cleavage occurs between the two nucleotides withrelease of the eTag reporter, with the terminal nucleotide of thedinucleotide labeled with a biotin (the eTag reporter would be releasedwithout the biotinylated nucleotide). In the case of a peptide analyte,one would have cleavage at a site, where the ligand remains with thepeptide analyte. For example, one could have the eTag reportersubstituted for the methyl group of methionine. Using the pyrazolone ofthe modified methionine, one could bond to an available lysine. Theamino group of the pyrazolone would be substituted with biotin. Cleavagewould then be achieved with cyanogen bromide, releasing the eTagreporter, but the biotin would remain with the peptide and any eTagreporter that was not released from the binding member. Avidin is thenused to change the polarity or sequester the eTag reporter conjugated tothe binding compound.

[0140] The separation of the eTag reporters by electrophoresis can beperformed in conventional ways. See, for example, U.S. Pat. Nos.5,750,015; 5,866,345; 5,935,401; 6,103,199, and 6,110,343 and WO98/5269,and references cited therein. Also, the sample can be prepared for massspectrometry in conventional ways. See, for example, U.S. Pat. Nos.5,965,363; 6,043,031; 6,057,543 and 6,111,251.

[0141] For convenience, kits can be provided comprising building blocksfor preparation of eTag reporters in situ or have assembled eTagreporters for direct bonding to the binding compound. For preparing theeTag reporter in situ during the synthesis of oligonucleotides, onewould provide phosphoramidites or phosphates, where the esters wouldinclude alkyl groups, particularly of from 1 to 3 carbon atoms, andcyanoethyl groups, while for the phosphoramidite, dialkylamino, wherethe alkyl groups are of from 1-4 carbon atoms, while the other groupwould be a protected hydroxy, where the protecting group would be commonto oligonucleotide synthesis, e.g. dimethoxytrityl. For large numbers ofeTag reporters, that is, 20 or more, one kit would supply at least 3each of mass-modifying regions and charge-modifying regions, each havingat least the phosphate linking group and a protected hydroxyl. The twofunctional groups may be separated by 2 or more atoms, usually not morethan about 60 atoms, and may be vicinal (α,β to α,ω). The nature of thecompounds has been discussed previously. In the simplest case, thephosphorous acid derivative would serve as the charge-modifying region,so that the mass-modifying region and the charge-modifying region wouldbe added as a single group. In addition, one would have at least 2detectable regions, which would be a fluorescer having the phosphatelinker and other functionalities protected for purposes of thesynthesis. Alternatively, instead of having the detection region theterminal region, where the detectable region allows for the presence oftwo functionalities that can be used for linking, one of the otherregions may serve as the terminal region. Also, one of the regions maybe conveniently linked to a mono- or dinucleotide for direct linking tothe oligonucleotide chain, where cleavage will occur at the 3′ site ofthe nucleotide attached to the eTag reporter. By using tri- ortetrasubstituted groups, one can provide a detectable region thatprovides the pair for energy transfer. One need only have one or twodifferent energy transfer agents, while having a plurality of emittingagents to greatly expand the number of different eTag reporters.

[0142] Other reagents that are useful include a ligand-modifiednucleotide and its receptor. Ligands and receptors include biotin andstrept/avidin, ligand and antiligand, e.g. digoxin or derivative thereofand antidigoxin, etc. By having a ligand conjugated to theoligonucleotide, one can sequester the eTag conjugated oligonucleotideprobe and its target with the receptor, remove unhybridized eTagreporter conjugated oligonucleotide and then release the bound eTagreporters or bind an oppositely charged receptor, so that theligand—receptor complex with the eTag reporter migrates in the oppositedirection.

[0143] Where one prepares the eTag reporter, there will be theadditional linking region, which in the above description is served bythe phosphorous acid derivative or the mono- or dinucleotide unitphosphorous acid derivative. For these eTag reporters, one need not berestricted to phosphate links, but may use other convenient chemistries,particularly chemistries that are automated. Thus, instead ofphosphorous acid and protected alcohol, one can use carboxy and alcoholor amino, activated olefin and thiol, amino and oxo-carbonyl,particularly with reductive amination, an hydroxy with an active halideor another hydroxy to form an ether, and the like. One may employcompounds that are difunctional with the same or differentfunctionalities, where one could have a diacid and a diol or anhydroxyacid or cyclic ester for producing the eTag reporter. Numerousexamples of these types of compounds have already been described and arewell known in the literature. By appropriate selection of the monomersand conditions, one can select a particular order of reaction, namelythe number of monomers that react or one may separate the mixture by thedifferent mobilities.

[0144] For separations based on sorption, adsorption and/or absorption,the nature of the eTag reporters to provide for differentiation can berelatively simple. By using differences in composition, such asaliphatic compounds, aromatic compounds and halo derivatives thereof,one may make the determinations with gas chromatography, with electroncapture or negative ion mass spectrometry, when electronegative atomsare present. In this way one may use hydrocarbons or halo-substitutedhydrocarbons as the eTag reporters bonded to a releasable linker. See,U.S. Pat. Nos. 5,565,324 and 6,001,579, which are specificallyincorporated by reference as to the relevant disclosure concerningcleavable groups and detectable groups.

[0145] The kits will include at least two detectable regions andsufficient reagents to have at least 10, usually at least 20 andfrequently at least 50 or more different eTag reporters that can beseparated by their mobility.

[0146] For 20 different eTag reporters, one only requires 5 differentmass-modifying regions, one phosphate link and four different detectableregions. For 120 eTag reporters, one need only have 10 differentmass-modifying regions, 3 different charge-modifying regions and 4different detectable regions. For 500 different eTag reporters, one needonly have 25 different mass-modifying regions, 5 differentcharge-modifying regions and 4 different detectable regions.

[0147] For an inclusive but not exclusive listing of the various mannersin which the subject invention may be used, the following table isprovided.

[0148] Recognition event leads to generation or modification of eTagreporters. eTag reporter Recognition Event Activation Amplification ModeFormat Binding Assays (solution Multiplexed assays (2- Phase eTagreporter generation 1000) leading to release followed by separation byCE, of library of eTag HPLC or Mass Spectra) reporters. Every eTagreporter codes for a unique binding event or assay. Hybridizationfollowed by 5′ Nuclease assay PCR, Invader Sequence recognition forenzyme recognition example for multiplexed gene expression, SNP'sscoring etc . . . 3′ Nuclease assay Multiplexed assays Sequencerecognition Restriction enzymes Multiplexed assays Sequence recognitionRibonuclease H Multiplexed assays Sequence recognition Hybridizationfollowed by Singlet Oxygen Single eTag reporter release Multiplexedassays channeling per binding event Sequence recognition Hybridizationfollowed by Singlet Oxygen Amplification due to turnover Multiplexedassays channeling of eTag reporter binding Sequence recognition moietyAmplification due to release Multiplexed assays of multiple eTagreporters Sequence recognition (10 to 100,000) per binding eventHydrogen peroxide Amplification due to turnover Multiplexed assays ofeTag reporter binding Sequence recognition moiety Amplification due torelease Multiplexed assays of multiple eTag reporters Sequencerecognition (10 to 100,000) per binding event Light; EnergyAmplification due to turnover Multiplexed assays Transfer(Photocleavage) of eTag Sequence recognition reporter binding moietyAmplification due to release Multiplexed assays of multiple eTagreporters Sequence recognition (10 to 100,000) per binding eventIMMUNOASSAYS Sandwich assays Singlet Oxygen A few (2-10) eTag reportersProteomics Antibody-1 decorated with release per binding eventMultiplexed Immunoassays Sensitizer while antibody-2 Is decorated withsinglet oxygen cleavable eTag reporters Singlet Oxygen Amplification dueto release Proteomics of multiple eTag reporters MultiplexedImmunoassays (10 to 100,000) per binding event Sandwich assays HydrogenPeroxide A few (2-10) eTag reporters Proteomics Antibody-1 decoratedwith release per binding event Multiplexed Immunoassays Glucose oxidasewhile antibody-2 Is decorated with hydrogen peroxide cleavable eTagreporters Hydrogen Peroxide Amplification due to release Proteomics ofmultiple eTag reporters Multiplexed Immunoassays (10 to 100,000) perbinding event Competition assays Singlet Oxygen A few (2-10) eTagreporters Antibody-1 decorated with release per binding event Sensitizerwhile Antigen Is decorated with singlet oxygen cleavable eTag reporters

[0149] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL

[0150] Synthetic Preparation of Modified Fluorescein Phosphoramidites

[0151] Pivaloyl protected carboxyfluorescein: Into a 50 mL round bottomflask was placed 5(6)-carboxyfluorescein (0.94 g, 2.5 mmol), potassiumcarbonate (1.0 g, 7.5 mmol) and 20 mL of dry DMF. The reaction wasstirred under nitrogen for 10 min, after which trimethylacetic anhydride(1.1 mL, 5.5 mmol) was added via syringe. The reaction was stirred atroom temperature overnight, and then filtered to remove excess potassiumcarbonate and finally poured into 50 mL of 10% HCl. A sticky yellowsolid precipitated out of solution. The aqueous solution was decantedoff and the residual solid was dissolved in 10 mL of methanol. Dropwiseaddition of this solution to 10% HCl yielded a fine yellow precipitate,which was filtered and air dried to yield an off white solid (0.88 g,62%). TLC (45:45:10 Hxn,EtOAc,MeOH)

[0152] NHS ester of protected pivaloyl carboxyfluorescein. Into a 200 mLround bottom flask was placed the protected carboxyfluorescein (2.77 g,5.1 mmol) and 50 mL of dichloromethane. N-hydroxysuccinimide (0.88 g,7.6 mmol) and dicyclohexylcarbodiimide (1.57 g, 7.6 mmol) were added andthe reaction was stirred at room temperature for 3 hours. The reactionwas then filtered to remove the precipitated dicyclohexyl urea byproductand reduced to approx. 10 mL of solvent in vacuo. Dropwise addition ofhexanes with cooling produced a yellow-orange colored solid, which wastriturated with hexanes, filtered and air dried to yield 3.17 g (95%) ofproduct. TLC (45:45:10 Hxn,EtOAc,MeOH)

[0153] Alcohol. Into a 100 mL round bottom flask was placed the NHSester (0.86 g, 1.34 mmol) and 25 mL of dichloromethane. The solution wasstirred under nitrogen after which aminoethanol (81 μL, 1 eq) was addedvia syringe. The reaction was monitored by TLC (45:45:10 Hxn,EtOAc,MeOH)and was found to be complete after 10 min. The dichloromethane was thenremoved in vacuo and the residue dissolved in EtOAc, filtered andabsorbed onto 1 g of silica gel. This was bedded onto a 50 g silicacolumn and eluted with Hxn:EtOAc:MeOH (9:9:1) to give 125 mg (20%) ofclean product.

[0154] Phosphoramidite. Into a 10 mL round bottom flask containing 125mg of the alcohol was added 5 mL of dichloromethane. Diisopropylethylamine (139 μl, 0.8 mmol) was added via syringe. The colorlesssolution turned bright yellow. 2-cyanoethyldiisopropylchlorophosphoramidite (81 μl, 0.34 mmol) was added viasyringe and the solution immediately went colorless. After 1 hour TLC(45:45:10 Hxn:EtOAc:TEA) showed the reaction was complete with theformation of two closely eluting isomers. Material was purified on asilica column (45:45:10 Hxn:EtOAc:TEA) isolating both isomers togetherand yielding 130 mg (85%).

[0155] Carboxylic acid. Into a 4 mL vial was placed 12-aminododecanoicacid (0.1 g, 0.5 mmol) and 2 mL of pyridine. To this suspension wasadded chlorotrimethyl silane (69 μL, 1.1 eq) via syringe. After allmaterial dissolved (10 min) NHS ester (210 mg, 0.66 eq) was added. Thereaction was stirred at room temperature overnight and then poured intowater to precipitate a yellow solid, which was filtered, washed withwater, and air dried. TLC (45:45:10 Hxn:EtOAc:MeOH) shows a mixture oftwo isomers.

[0156] General Procedure for Remaining Syntheses. The carboxylic acidformed described above is to be activated by NHS ester formation with1.5 eq each of N-hydroxysuccinimide and dicyclohexylcarbodiimide indichloromethane. After filtration of the resulting dicyclohexylurea,treatment with 1 eq of varying amino alcohols will effect amide bondformation and result in a terminal alcohol. Phosphitylation usingstandard conditions described above will provide the phosphoramidite.

Synthesis of Fluorescein Phosphoramidites

[0157]

[0158] Synthesis of 3′,5′-O-di-t-butyldimethylsilyl-2′-Deoxyuridine(1):

[0159] 2′-Deoxyuridine (4 gm, 17.5 mmol) and imidazole (3.47 gm, 52.5mmol) were dissolved in 30 ml of dry DMF and t-butyldimethyl-silylchloride (7.87 gm, 52.5 mmol) added to the stirring solution at roomtemperature. After 3 hrs, TLC on silica gel (10% MeOH+90% CH₂Cl₂) showedthat all starting material had been converted to a new compound withhigher R_(f). The solution was concentrated into a small volume, thenabout 200 ml of ether was added and washed three times with saturatedaqueous NaCl solution. The organic layer was dried over anhydrousNa₂SO₄, and the filtrate was evaporated to give a colorless gummymaterial which converted to a white solid product (eight gm, 100 %).This product was identified with HNMR and ES-MS.

[0160] Synthesis of3′,5′-O-di-t-butyldimethylsilyl-N⁴-(1,2,4-triazolo)-2′-Deoxycytidine(2):

[0161] 1,2,4-Triazole (19.45 gm, 282 mmol) was suspended in 300 ml ofanhydrous CH₃CN at 0° C., 8 ml of POCl₃, then 50 ml of trliethylaminewas added slowly in 5 min. After an hour,3′,5′-O-Di-t-butyldimethylsilyl-2′-Deoxyuridine (1) (9 gm, 19.7 mmol)was dissolved in 200 ml of dry CH₃CN and added to the reaction over 20min. After stirring the reaction for 16 hours at RT, TLC (100% ether)showed that all starting material was converted to a new compound withlower R_(f). The reaction mixture was filtered, reduced the volume ofCH₃CN, diluted with ethyl acetate and washed with saturated aqueousNaHCO₃ then twice with saturated aqueous NaCl. The organic layer wasdried over anhydrous Na₂SO₄ and the solvent was evaporated,co-evaporated from toluene to give a yellow solid product (10 gm. 100%).This product was identified with HNMR and ES-MS.

[0162] Synthesis of3′,5-O-di-t-butyldimethylsilyl-N⁴-(4,7,10-trioxa-1-tridecaneamino)-2′-Deoxycytidine(3):

[0163] 4,7,10-Trioxa-1,13-tridecanediamine (10.44 gm, 47.4 mmol) wasdissolved in 100 ml dioxane, then3′,5-O-di-t-butyldimethylsilyl4-(1,2,4-triazolo)-2-deoxycytidine (2)(8.03 gm, 15.8 mmol) was dissolved in 200 ml of dioxane (heated to about50 C. and cooling it dawn to RT) and added dropwise in 10 min., to thesolution of 4,7,10-Trioxa-1,13-tridecanediamine with vigorous stirringat RT. After 5 hrs, TLC on silica gel showed that all starting materialwas converted to a new product with lower R_(f), the resulting mixturewas evaporated to dryness. The residue was dissolved in dichloromethaneand washed twice with 5% sodium bicarbonate solution and saturatedsodium chloride solution. The organic layer was dried over sodiumsulphate, filtered and evaporated to dryness to give a yellow gummyproduct (7.87 gm). The product was purified on a silica gel columneluted with a gradient of 0 to 10% methanol in dichloromethane with 1%triethylamine. The product was obtained as a yellowish gum ( 5.66 gm,54%). This product was identified with HNMR and ES-MS.

[0164] Synthesis of3′,5′-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-Deoxycytidine(4):

[0165] 3 ′,5′-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecaneamino)-2′-deoxycytidine(3)(2.657gm, 4.43 mmol) and Biotin-NHS ester (1.814 gm,5.316 mmol) were dissolvedin 20 ml of dry DMF and about 1 ml of triethylamine was added. Afterstirring the reaction mixture for 4 hrs at RT, the reaction was stoppedby evaporating all DMF to give a yellow gum material (4.36 gm). Thismaterial was dissolved in dichloromethane and washed three times withsaturated solution of NaCl, dried over sodium sulphate and evaporated todryness. TLC on silica gel (5% MeOH+1% TEA+94% CH₂Cl₂) indicated theformation of a new product which was higher R_(f). This product waspurified with column chromatography on silica gel using (99% CH₂Cl₂+1%TEA) to (1% MeOH+1% TEA+98% CH₂Cl₂) to yield a yellow foamy product(2.13 gm, 60%). This product was identified with HNMR and ES-MS.

[0166] Synthesis of4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-Deoxycytidine(5):

[0167]3′,5′-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-deoxycytidine(4)(1.6 gm, 1.8 mmol) was dissolved in 50 ml of dry THF, then about 5.5 mlof tetrabutylammonium fluoride in THF was added in 2 min. while stirringat RT. After 3 hrs, TLC on silica gel (10% MeOH+1% TEA+89% CH₂Cl₂)showed that a new product with lower R_(f) formed. The solvent wasevaporated to give a yellow oily product. Column chromatography onsilica gel eluted with (99% CH₂Cl₂+1% TEA) to (7% MeOH+1% TEA+92%CH₂Cl₂) permitted the purification of the product as a gummy colorlessproduct (1.14 gm, 97%). This product was identified with HNMR and ES-MS.

[0168] t-Butylbenzoylation of the biotin of4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-deoxycytidine (6):

[0169] 4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-Deoxycytidine (5) (14.14 gm, 21.5 mmol) was dissolved in 100 ml of dry pyridine.Chlorotrimethyl silane (11.62 gm, 107.6 mmol) was added and the mixturewas stirred for 2 hrs at RT. 4-t-butylbenzoyl chloride (5.07 gm, 25.8mmol) was added and the mixture was stirred for another 2 hrs at RT. Thereaction mixture was cooled with ice-bath and the reaction stopped byadding 50 ml of water and 50 ml of 28% aqueous ammoni a solution. Thesolution kept stirring at RT for 20 min., then evaporated to dryness inhigh vacuum and finally co-evaporated twice from toluene. The materialwas dissolved in dichloromethane and extracted twice with 5% aqueoussodium bicarbonate solution. The organic layer was dried over sodiumsulphate, evaporated to dryness, re-dissolved in dichloromethane andapplied to a silica gel column. The column was eluted with gradient from0 to 10% of methanol in dichloromethane and obtained a product as awhite foam ( 9.4 gm, 53.5%). This product was identified with HNMR andES-MS.

[0170] Synthesis of5′-O-(4,4′-dimethoxytriphenylmethyl)-4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-Deoxycytidine(7):

[0171] Compound (6) (10.82 gm, 13.3 mmol) was co-evaporated twice fromdry pyridine, then dissolved in pyridine (100 ml) and4,4′-dimethoxytritylchloride(DMT-Cl) (6.76 gm, 19.95 mmol) was added andthe resulting mixture stirred for 3 hrs. TLC (10% MeOH+1% TEA+89%CH₂Cl₂) showed the formation of new product with higher R_(f), and somestarting material remained unreacted, then another amount of DMTCI (2gm) was added and kept stirring for 2 hrs. The reaction stopped byadding ethanol and the mixture was stirred for 15 min. After evaporationto dryness and co-evaporation from toluene, the material was dissolvedin dichloromethane. The organic layer washed twice with 5% aqueoussodium bicarbonate solution, dried over sodium sulphate, evaporated todryness. The product was purified on a silica column using a gradient ofmethanol from 0 to 5% in dichloromethane/1% TEA. The product wasobtained as a white foam (4.55 gm, 31%). This product was identifiedwith HNMR and ES-MS.

[0172] Synthesis of 3′-O-[(diisopropylamine)(2-cyanoethoxy)phosphino)]-5′-O-(4,4′-dimethoxytriphenylmethyl)-4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2′-Deoxycytidine(8):

[0173] The 5′-DMT-Biotin-dC (7) (507 mg, 0.453 mmol) was dissolved indry acetonitrile (30 ml) and dichloromethane (5 ml), thendiisopropylamine (73 mg, 0.56 mmol), tetrazole (1.15 ml, 0.52 mmol) and2-cyanoethyl N,N,N′N′-tetraisopropylphosphane 214 mg, 234 ul, 0.7 mmol)were added and the mixture stirred under nitrogen at RT. After 2 hrs,TLC on silica gel (45%/45%/5%/5%: Ethylacetate/dichloromethane/triethylamine/methanol) showed that only about30% of product was formed and about 70% of starting material wasunreacted. More reagents were added until most of starting material wasconverted, only about 5% left unreacted. The solvent was evaporated todryness, dissolved in dry dichloromethane, washed with sodiumbicarbonate solution (5%), saturated brine solution, then the organiclayer dried over sodium sulphate, evaporated to dryness. Columnchromatography on silica gel using (48%/48%/4%: Ethylacetate/dichloromethane/triethylamine) to (47%/47%/5%/1%: Ethylacetate/dichloromethane/triethylamine/methanol). The desired product wasobtained as a colorless gummy product (406 mg, 70%). This material wasco-evaporated three times from a mixture of dry benzene anddichloromethane, then was kept in desiccated containing P₂O₅ and NaOHpellets under vacuum for 26 hrs before used in DNA synthesis.

[0174] Synthesis of Biotinylated 2′-Deoxyadenosine Phosphoramidite;

[0175] Scheme#2.

[0176] Synthesis of 8-Bromo-2′-Deoxyadenosine:

[0177] 2′-Deoxyadenosine (7 gm. 25.9 mmol) was dissolved in sodiumacetate buffer (150, 1 M, pH5.0) by worming it to about 50 C., then wascooled dawn to 30 C., then 3 ml of bromine in 100 ml of the same bufferwas added dropwise at RT for 15 min., to the reaction. After, 6 hrs theTLC on silica gel (20% MeOH in CH2Cl2) showed that all starting materialwas converted to a new product. The reaction was discolored by addingsome sodium metabisulfite (Na₂S₂O₅) while it was stirring, the colorchanged to a white solution, the pH of the reaction was neutralized byadding NaOH (1M solution). The reaction mixture was kept at 4° C.(refrigerator) for 16 hrs. Next day the solid material was filtered,washed with cold water, then acetone to give a solid yellow powderproduct (5.75 gm. 64%). The structure of this product was confirmed by HNMR and ES-MS.

[0178] Synthesis ofN⁶-Benzoyl-8-bromo-5′-O-(4,4′-dimethoxytrityl)-2′-Deoxyadenosine (1):

[0179] 8-Bromo-2′-Deoxyadenosine (7.7 gm. 22.17 mmol) was dried byco-evaporation with dry pyridine and the solid was suspended in 200 mlof dry pyridine followed by the addition of4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl) (9 gm, 26.6 mmol). Afterstirring for 4 hrs at RT, TLC on silica gel showed that a new productwas formed and some starting material was unreacted. Another amount ofDMT-Cl (3 gm) was added and stirred at RT for 2 hrs. When TLC showedthat all starting material was converted to new product with higher Rf,the reaction mixture was cooled to 0 C. and trimethylchlorosilane(12.042 gm., 14 ml, 110.85 mmol) was added dropwise while cooling andafter 40 min. while stirring benzoyl chloride (15.58 gm, 12.88 ml,110.85 mmol) was similarly added. The reaction was allowed to react atRT over 2 hrs. The reaction was quenched by slow addition of cold water(50 ml), followed by addition of concentrated ammonia (30%, 50 ml).After 30 min. the reaction mixture was evaporated to dryness. Theresidue was dissolved in water, and the solution was extracted withethyl acetate three times, the organic layer washed with saturatedsodium bicarbonate solution, and then brine. The organic phase was driedover sodium sulphate, evaporated to dryness. The product was purified ona silica column chromatography, to give a yellowish solid product (6.79gm, 41.6%). The structure of this product was confirmed by H NMR andES-MS.

[0180] Synthesis ofN⁶-benzoyl-8-bromo-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine:

[0181]6N-Benzoyl-8-bromo-5′-O-(4,4′-dimethoxytrityl)-2′-Deoxyadenosine(1) (14gm. 19 mmol) and imidazole (1.94 gm, 28.5 mmol) were dissolved in 100 mlof dry DMF and t-butyldimethyl-silyl chloride (4.3 gm, 28.5 mmol) addedto the stirring solution at room temperature. After 4 hrs, TLC on silicagel (2.5% MeOH in CH₂Cl₂) showed that all starting material had beenconverted to a new product with higher R_(f). The solution wasconcentrated into a small volume, then about 400 ml of ether was addedand washed three times with saturated aqueous NaCl solution. The organiclayer was dried over anhydrous Na₂SO₄, and the filtrate was evaporatedto give an off-white foamy product (16.18 gm, 100%). H NMR and ES-MSconfirmed the structure.

[0182] Synthesis ofN⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneamino)-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(2):

[0183]N⁶-Benzoyl-8-bromo-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(8.31 gm. 9.7 mmol) was dissolved in 200 ml of ethanol then4,7,10-trioxa-1,13-tridecanediamine (6.75 gm. 6.7 ml. 30 mmol) was addedat once and kept stirring at 50 C. After 16 hrs TLC showed that allstarting material was converted to a one major product with lower Rf andother minor products. The solvent was evaporated to dryness, dissolvedin dichloromethane, washed three times with solution of brine, driedover anhydrous Na₂SO₄, evaporated to give a yellow gummy material.Column chromatography (1% TEA+CH₂Cl₂) to (1% TEA+5% MeOH+CH₂Cl₂)permitted the purification of the major product as an off-white gummymaterial (4.53 gm. 47%). This product was identified with HNMR andES-MS.

[0184] Synthesis ofN⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine:

[0185]N⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneamino)-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(4.53 gm. 4.57 mmol) and biotin-NHS ester (3.12 gm. 9.13 mmol) weredissolved in 75 ml of DMF and few drops of TEA were added and thereaction was stirred at RT. After, 2 hrs TLC on silica gel (5% MeOH+1%TEA+94 CH₂Cl₂) showed the formation of one major product less polar thanstarting material and another minor spot has lower Rf. The solvent wasevaporated to dryness, then dissolved in CH₂Cl₂ and washed three timeswith a saturated solution of NaCl, dried the organic layer, evaporatedto dryness to leave a yellow gummy material. This material was purifiedwith column chromatography on silica gel by using (1% TEA +CH₂Cl₂) to(1% TEA+2.5% MeOH+CH₂Cl₂) as eluant. After evaporating the fractionscontaining the product, gave a yellowish solid material (3.16 g, 78%).HNMR and ES-MS confirmed the structure.

[0186] Synthesis ofN⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(3):

[0187]N⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3′-O-t-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(3.16 gm, 2.6 mmol) was dissolved in 100 ml of dry THF, and then about(3.25 ml, 3.25 mmol) of tetrabutylammonium fluoride in THF was added in5 min. while stirring at RT. After 8 hrs, TLC on silica gel (10% MeOH+1%TEA+89% CH₂Cl₂) showed that a new product with lower R_(f) formed. Thesolvent was evaporated to give a yellow oily material. Columnchromatography on silica gel eluted with (99% CH₂Cl₂+1% TEA) to (5%MeOH+1% TEA+94% CH₂Cl₂) permitted the purification of the product as awhite foamy product (2.86 gm, 100%). HNMR and ES-MS confirmed thestructure.

[0188] Synthesis ofN⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3′-O-[(diisopropylamine)(2-cyanoethoxy)phosphino)]-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(4):

[0189]N⁶-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine(0.959 gm, 0.86 mmol) was dissolved in a mixture of dry acetonitrile(200 ml) and dichloromethane (50 ml), and diisopropylamine (224 ul, 1.29mmol) followed by the addition of 2-cyanoethyl N, N, N′,N′-tetraisopropylphosphane (404 ul, 1.29 mmol) and tetrazole (2.6 ml,1.2 mmol, 0.45 M solution in dry acetonitrile). The addition andsubsequent reaction are performed under argon while stirring at RT.After 1.5 h, TLC on silica gel (5% MeOH+5% TEA+45% EA+45% CH₂Cl₂) showedthat only about 50% of starting material (SM) was converted to a newproduct. The same above amount of reagents were added to the reactionand kept stirring for another 2 hrs at RT. TLC showed that about 95% ofSM was converted to a new product with higher R_(f). The solvent wasevaporated to dryness then was dissolved in dichloromethane, extractedonce with 5% solution of bicarbonate, followed by saturated brinesolution and then dried over anhydrous sodium sulfate and evaporated todryness. Column chromatography on silica gel (10% TEA+45% EA+45% CH₂Cl₂)first then (5% TEA+5% MeOH+45% EA+45% CH₂Cl₂). After evaporating thefractions containing the product, gave a yellow gummy material (774 mg).This material was co-evaporated three times from a mixture of drybenzene and dichloromethane, then was kept in desiccated containing P₂O₅and NaOH pellets under vacuum for 24 hrs before used in DNA synthesis.

[0190] Synthesis of oligonucleotides containing biotin-dC and Biotin-dA:

[0191] The syntheses of oligonucleotides containing biotin-dC andBiotin-dA, site-specifically located, were performed on a CPG supportusing a fully automated DNA synthesizer and the commercially availablefully protected deoxynucleosides phosphoramidites. Syntheses of allthese oligonucleotides were carried out at 1.0 and 0.4 μmol scale. Thecoupling time for the biotin-dC and dA were extended to 900 seconds. Thecoupling efficiency of the biotin-dC and dA phosphoramidites was foundgreater than 96%. After coupling of the biotinylated phosphoramidites,the remaining residues comprising the eTAG reporter of interest wereadded. Upon completion of the synthesis of the oligonucleotides, theywere deprotected with concentrated ammonia at 65° C. for 1 hour. Theseoligonucleotides were purified by reverse-phase HPLC and desalted by OPCcolumn, then used as such.

[0192] Synthetic Preparation of ACLA1 on an ABI 394 DNA Synthesizer

[0193] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by theaddition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram bottle ofthe fluorescein phosphoramidite, to give a 0.1M solution. The bottle isthen loaded onto the ABI 394 DNA synthesizer at position 8 using thestandard bottle change protocol. The other natural (dA^(bz)(0.1M: 0.25g/2.91 mL anhydrous acetonitrile), dC^(Ac)(0.1M: 0.25 g/3.24 mLanhydrous acetonitrile), dT(0.1M: 0.25 g/3.36 mL anhydrousacetonitrile), dG^(dmf)(0.1M: 0.25 g/2.81 mL anhydrous acetonitrile)phosphoramidite monomers are loaded in a similar fashion to ports 1-4.Acetonitrile is loaded onto side port 18, standard tetrazole activatoris loaded onto port 9, CAP A is loaded onto port 11, CAP B is loadedonto port 12, oxidant is loaded onto port 15, and deblock solution isloaded onto port 14 all using standard bottle change protocols.

[0194] Standard Reagents Employed for DNA Synthesis:

[0195] Oxidizer: 0.02 M Iodine (0.015 for MGB Probes)

[0196] DeBlock: 3% Trichloracetic Acid in Dichloromethane

[0197] Activator: 1H-Tetrazole in Anhydrous Acetonitrile

[0198] HPLC Grade Acetonitrile (0.002% water)

[0199] Cap A: Acetic Anhydride

[0200] Cap B: N-Methyl Imidazole.

[0201] The target sequence of interest is then input with a terminalcoupling from port 8 to attach ACLA1 to the 5′-end of the sequence. Amodified cycle is then chosen such that the desired scale (.2(mol,1.0(mol, . . . etc) of DNA is synthesized. The modified cycle containsan additional wait step of 800 seconds after any addition of 6-FAM. Astandard DNA synthesis column containing the support upon which the DNAwill be assembled is then loaded onto one of four positions of the DNAsynthesizer. DNA containing eTag reporters have been synthesized onvarious standard 500 Å CPG supports(Pac-dA-CPG,dmf-dG-CPG,Ac-dC-CPG,dT-CPG) as well as specialty supportscontaining 3′-biotin, 3′-amino linker, and minor grove binding species.

[0202] Upon completion of the synthesis, the column is removed from thesynthesizer and either dried under vacuum or by blowing air or nitrogenthrough the column to remove residual acetonitrile. The column is thenopened and the CPG is removed and placed in a 1-dram vial. Concentratedammonia is added (2.0 mL) and the vial is sealed and placed into a heatblock set at 65° C. for a minimum of two hours. After two hours the vialis allowed to cool to room temperature after which the ammonia solutionis removed using a Pasteur pipette and placed into a 1.5 mL Eppendorftube. The solution is concentrated in vacuo and submitted for HPLCpurification.

[0203] Synthetic Preparation of ACLA2 on an ABI 394 DNA Synthesizer

[0204] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by theaddition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram bottle ofthe fluorescein phosphoramidite, to give a 0.1M solution. The bottle isthen loaded onto the ABI 394 DNA synthesizer at position 8 using thestandard bottle change protocol. The other natural (dA^(bz)(0.1M: 0.25g/2.91 mL anhydrous acetonitrile), dC^(Ac)(0.1M: 0.25 g/3.24 mLanhydrous acetonitrile), dT(0.1M: 0.25 g/3.36 mL anhydrousacetonitrile), dG^(dmf) (0.1M: 0.25 g/2.81 mL anhydrous acetonitrile)phosphoramidite monomers are loaded in a similar fashion to ports 1-4.Acetonitrile is loaded onto side port 18, standard tetrazole activatoris loaded onto port 9, CAP A is loaded onto port 11, CAP B is loadedonto port 12, oxidant is loaded onto port 15, and deblock solution isloaded onto port 14 all using standard bottle change protocols. Thetarget sequence of interest is then input with a terminal coupling fromport 8 and a penultimate coupling of thymidine to the 5′-end of thesequence to assemble ACLA2. A modified cycle is then chosen such thatthe desired scale (.2 μmol, 1.0 μmol, . . . etc) of DNA is synthesized.The modified cycle contains an additional wait step of 800 seconds afterany addition of 6-FAM. A standard DNA synthesis column containing thesupport upon which the DNA will be assembled is then loaded onto one offour positions of the DNA synthesizer. DNA containing eTag reportershave been synthesized on various standard 500 Å CPG supports(Pac-dA-CPG,dmf-dG-CPG,Ac-dC-CPG,dT-CPG) as well as specialty supportscontaining 3′-biotin, 3′-amino linker, and minor grove binding species.

[0205] Upon completion of the synthesis the column is removed from thesynthesizer and either dried under vacuum or by blowing air or nitrogenthrough the column to remove residual acetonitrile. The column is thenopened and the CPG is removed and placed in a 1-dram vial. Concentratedammonia is added (2.0 mL) and the vial is sealed and placed into a heatblock set at 65° C. for a minimum of two hours. After two hours the vialis allowed to cool to room temperature after which the ammonia solutionis removed using a Pasteur pipette and placed into a 1.5 mL Eppendorftube. The solution is concentrated in vacuo and submitted for HPLCpurification.

[0206] Synthetic Preparation of ACLA3 on an ABI 394 DNA Synthesizer

[0207] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by theaddition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram bottle ofthe fluorescein phosphoramidite, to give a 0.1M solution. The bottle isthen loaded onto the ABI 394 DNA synthesizer at position 8 using thestandard bottle change protocol. The other natural (dA^(bz)(0.1M: 0.25g/2.91 mL anhydrous acetonitrile), dC^(Ac)(0.1M: 0.25 g/3.24 mLanhydrous acetonitrile), dT(0.1M: 0.25 g/3.36 mL anhydrousacetonitrile), dG^(dmf) (0.1M: 0.25 g/2.81 mL anhydrous acetonitrile)phosphoramidite monomers are loaded in a similar fashion to ports 1-4.Acetonitrile is loaded onto side port 18, standard tetrazole activatoris loaded onto port 9, CAP A is loaded onto port 11, CAP B is loadedonto port 12, oxidant is loaded onto port 15, and deblock solution isloaded onto port 14 all using standard bottle change protocols. Thetarget sequence of interest is then input with a terminal coupling fromport 8 and two penultimate couplings of thymidine to the 5′-end of thesequence to assemble ACLA3. A modified cycle is then chosen such thatthe desired scale (.2 umol, 1.0(mol, . . . etc) of DNA is synthesized.The modified cycle contains an additional wait step of 800 seconds afterany addition of 6-FAM. A standard DNA synthesis column containing thesupport upon which the DNA will be assembled is then loaded onto one offour positions of the DNA synthesizer. DNA containing eTags have beensynthesized on various standard 500 Å CPG supports(Pac-dA-CPG,dmf-dG-CPG,Ac-dC-CPG,dT-CPG) as well as specialty supportscontaining 3′-biotin, 3′-amino linker, and minor grove binding species.

[0208] Upon completion of the synthesis, the column is removed from thesynthesizer and either dried under vacuum or by blowing air or nitrogenthrough the column to remove residual acetonitrile. The column is thenopened and the CPG is removed and placed in a 1-dram vial. Concentratedammonia is added (2.0 mL) and the vial is sealed and placed into a heatblock set at 65° C. for a minimum of two hours. After two hours the vialis allowed to cool to room temperature after which the ammonia solutionis removed using a Pasteur pipet and placed into a 1.5 mL Eppendorftube. The solution is concentrated in vacuo and submitted for HPLCpurification.

[0209] Synthetic Preparation of ACLA16 on an ABI 394 DNA Synthesizer

[0210] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by theaddition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram bottle ofthe fluorescein phosphoramidite, to give a 0.1M solution. The bottle isthen loaded onto the ABI 394 DNA synthesizer at position 8 using thestandard bottle change protocol. Spacer phosphoramidite C3 (0.25 g) isdissolved in 5.0 mL of anhydrous acetonitrile and loaded onto position 5of the synthesizer. The other natural (dA^(bz)(0.1M: 0.25 g/2.91 mLanhydrous acetonitrile), dC^(Ac)(0.1M: 0.25 g/3.24 mL anhydrousacetonitrile), dT(0.1M: 0.25 g/3.36 mL anhydrous acetonitrile),dG^(dmf)(0.1M: 0.25 g/2.81 mL anhydrous acetonitrile) phosphoramiditemonomers are loaded in a similar fashion to ports 1-4. Acetonitrile isloaded onto side port 18, standard tetrazole activator is loaded ontoport 9, CAP A is loaded onto port 11, CAP B is loaded onto port 12,oxidant is loaded onto port 15, and deblock solution is loaded onto port14 all using standard bottle change protocols. The target sequence ofinterest is then input with a terminal coupling from port 8 and apenultimate coupling of the C3 spacer from port 5 to assemble ACLA16. Amodified cycle is then chosen such that the desired scale (.2 μmol, 1.0μmol, . . . etc) of DNA is synthesized. The modified cycle contains anadditional wait step of 800 seconds after any addition of 6-FAM. Astandard DNA synthesis column containing the support upon which the DNAwill be assembled is then loaded onto one of four positions of the DNAsynthesizer. DNA containing eTag reporters have been synthesized onvarious standard 500 Å CPG supports (Pac-dA-CPG,dmf-dG-CPG,Ac-dC-CPG,dT-CPG) as well as specialty supports containing 3′-biotin, 3′-aminolinker, and minor grove binding species.

[0211] Upon completion of the synthesis the column is removed from thesynthesizer and either dried under vacuum or by blowing air or nitrogenthrough the column to remove residual acetonitrile. The column is thenopened and the CPG is removed and placed in a 1-dram vial. Concentratedammonia is added (2.0 mL) and the vial is sealed and placed into a heatblock set at 65° C. for a minimum of two hours. After two hours the vialis allowed to cool to room temperature after which the ammonia solutionis removed using a Pasteur pipette and placed into a 1.5 mL Eppendorftube. The solution is concentrated in vacuo and submitted for HPLCpurification.

[0212] All other eTag reporters are synthesized in a similar manner tothat described above.

[0213] FIGS. 2A-2I provide a list of different eTag reporters with theirstructures, where the symbols are as defined in Table 2 and are repeatedhere for convenience. C₃, C₆, C₉ and C₁₈ are commercially availablephosphoramidite spacers from Glen Research, Sterling, Va. The units arederivatives of N,N-diisopropyl, O-cyanoethyl phosphoramidite, which isindicated by Q. The subscripts indicate the number of atoms in thechain, which comprises units of ethyleneoxy terminating in Q with theother terminus protected with DMT. The letters without subscripts A, T,C and G indicate the conventional nucleotides, while_(T) ^(NH) ^(₂)intends amino thymidine and C^(Br) intends bromocytidine. In FIG. 9, thenumbers indicate the eTag reporter as numbered in FIGS. 2A-2I.

[0214] S1 Nuclease Digestion of eTag Reporter Probes

[0215] In a 1.5 ml tube, add 10 μl of eTag reporter probe at aconcentration of 10 μM, add 1.5 μl of 10×S1 nuclease reaction buffer,add 0.5 μl of S1 nuclease (Promega, Cat#M5761, 20-100 unit/μl), and add3 μl of Tris-EDTA buffer to bring the final volume to 15 μl. Incubatethe reaction at 37° C. for 20 min followed by 25 min at 96° C. toinactivate the nuclease. Elution Time on 3100 E-Tag POP 4 (min)

6.85

8.06

8.05

6.43

6.57

7.02

6.90

7.49

5.81

9.15

6.43

4.72

6.15

3.82

4.55

4.26

4.45

3.51

2.98

4.50

[0216] TABLE 6

8.45

[0217] 5′Nuclease Assays for Monitoring Specific mRNA Expression in CellLysates

[0218] THP-1 cells (American Type Culture Collection, Manassas, Va.)were cultured in the presence or absence of 10 nM phorbol 12-myristate13-acetate (Sigma-Aldrich, St. Louis, Mo.) in RPMI 1640 medium with 10%fetal bovine serum (v/v), 2 mM L-glutamine, 10 mM HEPES, 0.05 mM2-mercaptoethanl. Twenty-four hours after the induction, cells wereharvested and washed twice with PBS before lysed with lysis buffer (20nM Tris pH7.5, 0.5% Nonidet P-40, 5 mM MgCl2, 20 ng/ul tRNA) at 25° C.,for 5 min. The lysate was heated at 75° C. for 15 min before tested in5′ nuclease assay.

[0219] Ten microliter cell lysate was combined with a single strandedupstream invader DNA oligo, (5′CTC-TCA-GTT-CT) (SEQ ID NO: 1), a singlestranded downstream biotinylated signal DNA oligo (eTag-labeled), and 2ng/ul 5′ nuclease (Cleavase IX) in 20 ul of buffer (10 mM MOPS pH 7.5,0.05% Tween-20 and 0.05% Nonidet P-40, 12.5 mM MgCl2, 100 uM ATP, 2 U/ulRnase inhibitor). The reactions were carried out at 60° C. for 4 hoursbefore analyzed by capillary electrophoresis. To eliminate backgroundsignal, due to the non-specific activity of the enzyme, 1 ul of 1 mg/mlavidin was added to the reactions to remove all the eTag-labeleduncleaved oligo, or eTag-labeled non-specifically cleaved oligos. FIGS.8 and 9 show separations that were conducted both with and without theaddition of avidin.

[0220] PCR Amplification with 5′ Nuclease Activity Using eTag Reporters

[0221] The eTag reporters are described in FIGS. 2A-2I. The eTagreporters that were prepared were screened to provide 20 candidates thatprovided sharp separations. 31 eTag reporters were generated withsynthetic targets using the TaqMan( reagents under conditions as shownin the following tabular format. There were 62 reactions with thesynthetic targets (1 reaction and one negative control for eTagreporter). The master mix involves preparing a solution of TaqMan mastermix, primer (both reverse and forward) and water. This mix is thenaliquoted into individual PCR tubes followed by the addition of probeand template. Volume (1 Stock Stock Conc. (25(μl/reax) Final conc.Master mix (Vol*64) TagMan mix 2×  6.25 0.5× 400 Probe (eTag 4 μM  1.25200 nM reporter) Primer 5 μM  2.5 500 nM 160 Template 100 fM  1.25 5 fmWater 13.75 880 Total 25 μl 1440/64 = 22.5 (+1.25 μl (probe) +  1.25 μl(template) =    25 μl.reax

[0222] All the individual reactions were then run on an ABI 3100 usingPOP4 as the separation matrix. The samples were diluted 1:20 in 0.5×TaqMan buffer and 1(1 of avidin (10 mg/ml) was added to bind to anyintact probe. The sample was further diluted 1:2 with formamide beforeinjecting the sample into the ABI 3100 capillaries. The following on theconditions used with the ABI 3100 for the separation. Temperature  60(C. Pre-run voltage  15 KV Pre-run time 180 sec Matrix POP4 Injectionvoltage  3 KV Injection time  10 sec Run voltage  15 KV Run time 900 secRun module eTag reporter POP4 Dye set D

[0223] Subsequent separation of multiple eTAG reporters in a single runwere accomplished as shown in FIG. 9, the structures of which areidentified in FIGS. 2A-2I.

[0224] eTag Reporter Proteomic Analog Assay

[0225] 1-Labeling of Aminodextran (MW ˜500,000) with eTag Reporter andBiotin.

[0226] Aminodextran was used as a model for demonstrating eTag reporterrelease in relation to a high molecular weight molecule, which alsoserves as a model for proteins. The number of amino groups for 10 mgaminodextran was calculated as 2×10⁻⁸ moles. For a ratio of 1:4 biotinto eTag reporter, the number of moles of biotin NHS ester employed was1.85×10⁻⁶ and the number of moles of maleimide NHS ester was 7.4×10⁻⁶.10.9 mg of aminodextran was dissolved in 6 ml of 0.1% PBS buffer. Then,10 mg of Biotin-x-x NHS ester and 23.7 mg of EMCS were dissolvedtogether in 1 ml of DMF. This DMF solution was added in 50 μl portion(30 min interval) to the aminodextran solution while it was stirring andkeeping away from the light. After final addition of the DMF solution,the mixtured was kept overnight (while stirring and away from thelight). Then, the mixture was dialyzed using membrane with cut offmolecular weight of 10,000. The membrane immersed in a beaker containing2 l of water while stirring. This water was changed four times (2 hinterval). The membrane was kept in the water overnight (while stirringand keeping away from the light). Then the solution was lyophilized andthe lyophilized powder was used for eTag reporter labeling.

[0227] 2-Reaction of Biotin and Maleimide Labeled Aminodextran with theeTag Reporter, SAMSA.

[0228] SAMSA[5-((2-(and-3)-S-acetylmercapto)succinoyl)amino)fluorescein]was employedas an eTag reporter to react with maleimide in the aminodextranmolecule. For this purpose 0.3 mg (˜5.3×10⁻⁹ moles) of biotin and EMCSlabeled with aminodextran were dissolved in 10 μl of water and thenreacted with 10 times the mol ratio of SAMSA, for the completeconversion of the maleimide to the eTag reporter. Therefore, 1.1 mg ofSAMSA (˜1.2×10⁻⁶ moles) is dissolved in 120 ∥l of 0.1 M NaOH andincubated at room temperature for 15 min (for the activation of thethiol group). Then the excess of NaOH was neutralized by the addition of2 μl of 6M HCl, and the pH of the solution was adjusted to 7.0 by theaddition of 30 μl of phosphate buffer (200 mM, pH=7.0). The activatedSAMSA solution was added to the 10 μl solution of the labeledaminodextran and incubated for 1 h. The eTag reporter labeledaminodextran was purified with gel filtration using Sephadex G-25(Amersham), and purified samples were collected.

[0229] 3-The Release of eTag from Labeled Aminodextran.

[0230] 2 μl of streptavidin coated sensitizer beads (100 μg/ml) wereadded carefully in the dark to the 5 μl of purified labeled aminodextranand incubated in the dark for 15 min. Then the solution was irradiatedfor 1 min at 680 nm. The release of the eTag reporter was examined be CEusing CE² LabCard™ device. As shown in FIG. 3, the CE² LabCard consistsof two parts; evaporation control and injection/separation. Theevaporation control incorporates a channel (450 μm wide and 50 μm deep)with two buffer reservoirs (2 mm in diameter) and the evaporationcontrol well (1 mm diameter) right in the center of the channel. Thevolume of the side wells (replenishment wells) are 4.7 μl while thevolume of the middle well is only 1.2 μl and the volume of the channelbeneath the middle well is about 40 nl. The second part of the CE²device which is the injection/separation part consists of injection andseparation channels with dimensions of 120 μm wide and 50 μm deep. Theinjection channel is connected directly to the evaporation control well.The channels are closed by laminating a film (MT40) on the LabCard™.

[0231] After filling the CE² LabCard device with the separation buffer(20 mM HEPES,pH=7.4 and 0.5% PEO), 300 nl of the assay mixture was addedto the middle well (sample well) and separated by CE as is shown in FIG.3.

[0232]FIG. 4 shows the electropherograms of purified labeledaminodextran with and without sensitizer beads. As shown, the additionof the sensitizer beads lead to the release of the eTag reporter fromthe aminodextran using singlet oxygen produced by sensitizer upon theirradiation at 680 nm. In order to optimize the irradiation time,different tubes containing the same mixture of beads and sensitizer wereirradiated for different lengths of time ranging from 1 to 10 min. Thereis no significant increase in the eTag reporter release for irradiationlonger than 1 min. FIG. 6, shows the effect of sensitizer beadconcentration on the eTag reporter release. As depicted in FIG. 6, thehigher concentration of sensitizer beads leads to the higher release ofeTag reporters from the labeled aminodextran. FIG. 7 depicts the linearcalibration curve for the release of eTag reporters as a function of thesensitizer bead concentration. In addition, the effect of theconcentration of labeled aminodextran on the eTag reporter release wasalso examined and the result is shown in FIG. 8. As can be seen, thelower concentration of labeled aminodextran for a given concentration ofsensitizer beads leads to more efficient eTag reporter release (orhigher ratio of eTag reporter released to the amount of labeledaminodextran).

[0233] It is evident from the above results that the subject inventionsprovide powerful ways of preparing compositions for use in multiplexeddeterminations and for performing multiplexed determinations. Themethods provide for homogeneous and heterogeneous protocols, both withnucleic acids and proteins, as exemplary of other classes of compounds.In the nucleic acid determinations, snp determinations are greatlysimplified where the protocol can be performed in only one to fourvessels and a large number of snps readily determined within a shortperiod of time with great efficiency and accuracy. For other sequences,genomes can be investigated from both prokaryotes and eukaryotes,including for the prokaryotes, drug resistance, species, strain, etc.and for the eukaryotes, species, cell type, response to externalstimuli, e.g. drugs, physical changes in environment, etc., mutations,chiasmas, etc. With proteins, one can determine the response of the hostcell, organelles or the like to changes in the chemical and physicalenvironments in relation to a plurality of pathways, changes in thesurface protein population, changes due to aging, neoplasia, activation,or other naturally occurring phenomenon, where the amount of protein canbe quantitated.

[0234] Particularly as to nucleic acid determinations, the subject eTagreporters can be synthesized conveniently along with the synthesis ofthe oligonucleotides used as probes, primers, etc., where the eTagreporter is released in the presence of the homologous target sequence.Kits of building blocks or eTag reporters are provided for use in thedifferent determinations.

[0235] All publications and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All publications and patentapplications set forth herein are incorporated by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporate by reference.

[0236] The invention now having been fully described, it will beapparent to one of ordinary skill in the art that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

1 1 1 11 DNA Human 1 ctctcagttc t 11

What is claimed is:
 1. A method for preparing a labeled oligonucleotideas a member of a family of labeled oligonucleotides each having adifferent mobility, said method comprising: synthesizing anoligonucleotide using an automated synthesizer employing a solidsurface; at the terminus of said synthesized oligonucleotide while boundto said surface sequentially adding at least two of a mass-modifyingregion, a charge-modifying region and a detectable region, using theautomated synthesizer where two of said regions can be combined in asingle region; to produce one member of a family of labeledoligonucleotides.
 2. A method according to claim 1, wherein saidoligonucleotides and regions are joined by phosphate linkages.
 3. Amethod according to claim 2, wherein said synthesizing and addingemploys phosphoramidites for producing said member.
 4. A method forpreparing a labeled oligonucleotide as a member of a family of labeledoligonucleotides each having a different mobility, said methodcomprising: synthesizing an oligonucleotide using an automatedsynthesizer employing a solid surface; at the terminus of saidsynthesized oligonucleotide while bound to said surface sequentiallyadding a mass-modifying region, a charge-modifying region and adetectable region, using the automated synthesizer where two of saidregions can be combined in a single region; wherein said mass-modifyinggroup is a neutral molecule, said charge-modifying region comprises atleast one amino acid, at least one carboxyl substituted polyester, or atleast one phosphate ester, and said detectable region is a fluorescer.to produce one member of a family of labeled oligonucleotides
 5. Amethod according to claim 4, wherein said mass-modifying group is ahydocarbon, a halohydrocarbon, a substituted aromatic compound, orcomprises at least one alkyleneoxy.
 6. A method according to claim 4,wherein said charge-modifying group comprises amino acids.
 7. A methodaccording to claim 4, wherein said charge-modifying region comprises aphosphate group and said regions are joined by phosphate groups, whereinsaid phosphate groups comprise said charge-modifying region.
 8. A methodaccording to claim 4, wherein said method is repeated at least 5 timesto produce 5 different labeled oligonucleotides.
 9. A method forpreparing a family of labeled oligonucleotides as members of a family oflabeled oligonucleotides each having a different mobility, said methodcomprising: synthesizing an oligonucleotide using an automatedsynthesizer employing a solid surface and phosphoramidite chemistry; atthe terminus of said synthesized oligonucleotide while bound to saidsurface sequentially adding a mass-modifying region, a charge-modifyingregion and a detectable region, using the automated synthesizer andphosphoramidite chemistry, where two of said regions can be combined ina single region; wherein said mass-modifying region is a neutral group,said charge-modifying region comprises at least one amino acid, at leastone carboxyl substituted polyester, or at least one phosphate ester, andsaid detectable region is a fluorescer; to produce one member of afamily of labeled oligonucleotides, and repeating said synthesizing andadding to produce additional members of said family.
 10. A methodaccording to claim 9, wherein said charge-modifying region comprises aphosphate.
 11. A method according to claim 9, wherein said neutral groupis an alkylene.
 12. A method according to claim 9, wherein said neutralgroup is an oxyalkylene.
 13. A compound comprising an oligonucleotideand in any order a mass-modifying region, a charge-modifying region anda detectable region joined by phosphate linkages.
 14. A compoundaccording to claim 13, where said phosphate linkages comprise saidcharge-modifying region and said neutral region is an alkylene, analkyleneoxy or a substituted aromatic group.
 15. A compound according toclaim 13, wherein said detectable region is a fluorescer.
 16. A compoundaccording to claim 15, wherein said fluorescer is fluorescein, afluorescein derivative, rhodamine, a rhodamine derivative, Cy-3 or Cy-5.17. A method of performing a multiplexed assay for the determination ofa plurality of target species in a sample employing eTag reporterconjugated binding compounds, wherein said eTag reporters are linked tosaid binding compounds by a cleavable linkage, are specific for thebinding compound to which said eTag reporter is conjugated, are otherthan oligonucleotides of at least 3 nucleotides and have at least onecharacteristic for individual detection, and said binding compounds areindividually specific for different target species, said methodcomprising: combining said sample with said compounds for said targetspecies under conditions for binding of said binding compounds to saidtarget species; releasing eTag reporters from eTag reporter conjugatedbinding compounds bound to said target species by cleavage of saidcleavable linkage; and identifying said released eTag reporters by meansof said at least one characteristic; whereby the presence of said targetcompounds in said sample is determined.
 18. A method according to claim17, wherein said target species are nucleic acid sequences homologous tosaid binding compounds.
 19. A method according to claim 18, wherein saidreleasing is by cleavage of a phosphate bond.
 20. A method according toclaim 17, wherein said target species are poly(amino acids).
 21. Amethod according to claim 20, wherein said cleavable linkage isphotolytically labile.
 22. A method according to claim 20, wherein saidreleasing results from an active agent in proximity to said cleavablelinkage.
 23. A method according to claim 17, wherein said at least onecharacteristic is mobility in an electrical field.
 24. A methodaccording to claim 17, wherein said at least one characteristic is massfor identification in a mass spectrometer.
 25. A method of performing amultiplexed assay for the determination of a plurality of nucleic acidtarget species in a sample employing eTag reporter substitutedoligonucleotides, wherein said oligonucleotides are homologous to saidtarget species and wherein said eTag reporters are specific for saidoligonucleotides to which said eTag reporter is substituted, are otherthan oligonucleotides of at least 3 nucleotides and have at least onecharacteristic for individual detection, said method comprising:combining said sample with said oligonucleotides for said target speciesunder conditions for hybridization of said oligonucleotides to saidtarget species; releasing eTag reporters from eTag reporter conjugatedoligonucleotides bound to said target species by cleavage of a phosphatebond of said oligonucleotide; and identifying said released eTagreporters by means of said at least one characteristic; whereby thepresence of said target compounds in said sample is determined.
 26. Amethod according to claim 25, wherein said cleavage of said phosphatebond releases said eTag reporter comprising one nucleotide.
 27. A methodaccording to claim 25, wherein said releasing includes a cleavase.
 28. Amethod according to claim 25, wherein said releasing includes apolymerase having nuclease activity.
 29. A method according to claim 25,wherein said releasing comprises as an additional step adding a primerto said sample homologous to a first portion of said target species 5′of a second portion of said target species to which said oligonucleotideis homologous.
 30. A method according to claim 25, wherein saididentifying comprises separating said released eTag reporters in anelectrical field.
 31. A method according to claim 25, wherein saididentifying comprises separating said released eTag reporters in amagnetic field.
 32. A method of performing a multiplexed assay for thedetermination of a plurality of poly(amino acid) target species in asample employing eTag reporter conjugated binding compounds, whereinsaid eTag reporters are linked to said binding compounds by a cleavablelinkage, are specific for the binding compound to which said eTagreporter is conjugated, are other than oligonucleotides of at least 3nucleotides and have at least one characteristic for individualdetection, and said binding compounds are individually specific fordifferent target species, said method comprising: combining said samplewith said compounds for said target species under conditions for bindingof said binding compounds to said target species; releasing eTagreporters from eTag reporter conjugated binding compounds bound to saidtarget species by cleavage of said cleavable linkage; and identifyingsaid released eTag reporters by means of said at least onecharacteristic; whereby the presence of said target compounds in saidsample is determined.
 33. A method according to claim 32, wherein saidcleavable linkage is photolytically labile.
 34. A method according toclaim 32, wherein said releasing comprises bringing said cleavablelinkage in proximity to an active agent.
 35. A method according to claim34, wherein said active agent is singlet oxygen.
 36. A method accordingto claim 34, wherein said active agent is produced at a solid support.37. A method according to claim 34, wherein said active agent isproduced by a second binding compound bound to said target species. 38.A method according to claim 32, wherein said binding compounds areantibodies or fragments thereof.
 39. A method according to claim 32.wherein said identifying comprises separation in an, electric field. 40.A method according to claim 32, wherein said separation comprisesseparation in a magnetic field.
 41. A method of performing a multiplexedassay for the determination of a plurality of target species in a sampleemploying eTag reporter conjugated binding compounds, wherein said eTagreporters are linked to said binding compounds by a cleavable linkage,are specific for the binding compound to which said eTag reporter isconjugated, are other than oligonucleotides of at least 3 nucleotidesand have at least one characteristic for individual detection, andwherein said eTag reporter conjugated binding compound comprises aligand bound at a site where said ligand remains with said bindingcompound upon cleavage of said cleavable linkage, said methodcomprising: combining said sample with said compounds for said targetspecies under conditions for binding of said binding compounds to saidtarget species; releasing eTag reporters from eTag reporter conjugatedbinding compounds bound to said target species by cleavage of saidcleavable linkage; adding a reciprocal binding member to said bindingcompound to bind to said eTag reporter conjugated binding compounds toreduce interfence with said determination; and identifying said releasedeTag reporters by means of said at least one characteristic; whereby thepresence of said target compounds in said sample is determined.
 42. Amethod according to claim 41, wherein said identifying is by separatingin an electrical field and said reciprocal binding member has theopposite polarity of said eTag reporters.
 43. A method according toclaim 42, wherein said eTag reporters are negatively charged and saidreciprocal binding member is positively charged.
 44. A method accordingto claim 43, wherein said ligand is biotin and said reciprocal bindingmember is streptavidin.
 45. A method for determining the change in thesurface membrane protein population for a plurality of surface membraneproteins by performing a multiplexed assay for the determination of saidplurality of surface membrane proteins of at least one cell in acellular sample by employing eTag reporter conjugated binding compounds,wherein said eTag reporters are linked to said binding compounds by acleavable linkage, are specific for the binding compound to which saideTag reporter is conjugated, are other than oligonucleotides of at least3 nucleotides and have at least one characteristic for individualdetection, said method comprising: combining said cellular sample withsaid compounds for said proteins under conditions for binding of saidbinding compounds to said proteins; releasing eTag reporters from eTagreporter conjugated binding compounds bound to said proteins by cleavageof said cleavable linkage; and identifying said released eTag reportersby means of said at least one characteristic; whereby the presence ofsaid proteins in said sample is determined.
 46. A method according toclaim 45, wherein said binding compounds consist of at least one ofligands for said surface membrane proteins and antibodies to saidsurface membrane proteins.
 47. A method according to claim 45, whereinsaid combining includes the addition of second binding compoundsconjugated with an active agent producing moiety, wherein said activeagent causes cleavage of said cleavable linkage.
 48. A method accordingto claim 47, wherein said active agent is singlet oxygen.